NOVEL POLYPEPTIDE HAVING FACTOR XA INHIBITORY ACTIVITY

Abstract: A novel polypeptide derived from the leech Hirudo medicinalis was found to be able to inhibit Factor Xa thereby reducing the extent of blood coagulation. The polypeptide is useful in treating conditions of excessive blood coagulation. A recombinant organism containing cDNA encoding the polypeptide has been deposited in the ATCC under Accession No. 69134. Recombinant microorganisms able to produce the polypeptide have been deposited in the ATCC under Accession Nos. 69135, 69137 and 69269.

Description: NOVEL POLYPEPTIDE HAVING FACTOR XA INHIBITORY ACTIVITY Background of the Invention Haemostasis. is the interaction of a complex series of processes aimed at ensuring the proper flow of blood through the closed, high-pressure circulatory system which comprises the mammalian vascular system. One aspect of haemostasis is the coagulation cascade which assists in maintaining the integrity of blood vessels following injury. The coagulation cascade is a complex series of processes which culminate in the formation of a fibrin clot at the site of blood vessel injury. Abnormal formation of blood clots is the cause of pathological conditions such as thrombi and embolisms. The coagulation cascade may be initiated either through the intrinsic pathway, in which all the protein components are present in blood, or through the extrinsic pathway, in which tissue factor, a cell membrane protein, plays a critical role. The last two steps of the coagulation cascade leading to clot formation are common to both pathways. The first of these two steps comprises the formation of the "prothrombinase complex" composed of prothrombin (the zymogen of thrombin) , Factor Xa (FXa) , and Factor Va on the platelet's membrane surface. Fxa is the enzyme which catalyzes the conversion of prothrombin to thrombin. Thrombin subsequently catalyzes the conversion of fibrinogen to fibrin, an insoluble polymer which is a major component of blood clots. Treatment with anticoagulants is indicated in a wide range of clinical situations such as thrombosis e.g. deep venous thrombosis (DVT) , disseminated intravascular coagulation (DIC) , and cardiovascular and cerebrovascular diseases. Other indications include pathophysiological conditions associated with post operative trauma, obesity, pregnancy, side effects of oral contraceptives, prolonged immobilization particularly in the aged, and other known clinical situations involving blood coagulation and thrombosis. Various references are noted by Arabic numbers in parentheses. These references are listed in numerical order at the end of the specification before the claims and are hereby incorporated by reference in their entirety to further explain the state of the art relevant to this application. The use of anticoagulants can be beneficial in treatment of both venous thrombosis (14) such as occurs in DVT and DIC, and arterial thrombosis such as occurs during reocclusion following thrombolysis (15) . The use of anticoagulants in acute coronary thrombosis is based on the established fact that the coagulation cascade is the primary cause of thrombogenicity also within platelet-rich arterial thrombi (16). In pathological conditions of excessive clot formation, coagulation can be inhibited either by blocking the catalytic activity of thrombin by heparin - whose action is mediated by the plasmatic inhibitor antithrombin III - or hirudin, or alternatively by inhibiting an earlier step of the coagulation cascade. For example, heparinoids (low molecular weight derivatives of heparin) are known to be selective inhibitors of the step preceding thrombin, i.e., they preferentially enhance the binding of antithrombin III to FXa, thus inhibiting the FXa-catalyzed conversion of prothrombin to thrombin. Since the blood concentration of Factor X is approximately 10-fold lower than that of prothrombin, much smaller amounts of FXa inhibitors than thrombin inhibitors are required to inhibit coagulation. Furthermore, FXa usually resides in the prothrombinase complex and its activity would have to be inhibited in that complex. However, the inhibition by complexes of antithrombin with these heparin derivatives appears to be effective only upon free FXa in the plasma and inefficient when FXa is incorporated in the prothrombinase complex, which is the location of FXa during thrombus formation. This is similar to the disclosure that FXa in a prothrombinase complex is inaccessible to inhibition by the heparin-antithrombin III conjugate (6) . At present, heparin is the most widely used anticoagulant and anti-thrombotic drug, but it has two disadvantages: firstly, it acts at the level of thrombin inhibition, thus necessitating the administration of relatively large amounts of inhibitor; and secondly, it is likely to cause excessive bleeding due to the systemic inhibition of thrombin which is required for normal hemostasis (7) . The use of hirudin and its low molecular weight analogs (hirulogs) probably entails similar disadvantages. These disadvantages prompted the search for new anticoagulant and anti-thrombotic substances suitable for therapeutic use. The use of a selective inhibitor of FXa. as an anticoagulant may reduce the problem of bleeding caused by the currently used anti-thrombotic drugs, such as heparin and hirudin and their analogs. This postulated advantage is due to the fact that a FXa inhibitor acts as a modulator of coagulation since it does not affect already existing thrombin and therefore does not completely neutralize normal hemostasis. This is because the existing thrombin is entrapped in active form in fibrin clots and is released during thrombolysis (17) . Two closely related factor Xa inhibitors have been isolated from the Mexican leech Haementeria officinalis (antistasin - references 1 and 2) and from the giant Amazonian leech Haementeria ghiliani (3, 4). A third FXa inhibitor - termed tick anti-coagulant peptide (TAP) isolated from the tick Ornithodorous moubata (5,9) has been cloned, expressed, purified and characterized (10). A fourth potent FXa inhibitor, isolated from the black fly, Simulium vittatum, has also been characterized (11) . Both in vitro and in vivo studies have shown that inhibition of FXa-mediated coagulation with two of these inhibitors, antistasin and TAP, is as effective as heparin in preventing venous thrombosis (12) . Additionally, Rigbi et al. (13) disclose a factor Xa inhibitor isolated from the saliva of the European leech Hirudo medicinalis. This application discloses the surprising and unexpected discovery of a novel Factor Xa inhibitor ("FXal") having a sequence different from that of any known FXa inhibitor. Si-urcmary of the Invention "FXal" as used herein refers to a novel polypeptide having FXa inhibitory activity which is present in the European leech Hirudo medicinalis and has a sequence different from that of any known FXa inhibitor. "Recombinant FXal", "recFXal", and "rFXal" all refer to the novel polypeptide having FXa inhibitory activity expressed by recombinant DNA; the term "FXal" is also often used to refer to recombinant FXal where no possibility of ambiguity exists. The present invention provides a polypeptide comprising the amino acid sequence X - Y - CYS GLN GLU GLU GLU CYS PRO ASP PRO TYR LEU CYS SER PRO VAL THR ASN ARG CYS GLU CYS THR PRO VAL LEU CYS ARG MET TYR CYS LYS PHE TRP ALA LYS ASP GLU LYS GLY CYS GLU ILE CYS LYS CYS GLU GLU ' LEU CYS GLN ASN GLN ASN CYS THR LYS ASP MET LEU CYS SER SER VAL THR ASN ARG CYS ASP CYS GLN ASP PHE LYS CYS PRO GLN SER TYR CYS - Z wherein X is MET or absent; Y is 0-29 amino acids of the sequence LYS MET CYS TRP ASN LYS GLY CYS PRO CYS GLY GLN ARG CYS ASN LEU HIS ARG ASN GLU CYS GLU VAL ILE ALA GLU ASN ILE GLU, with the proviso that if part of the sequence is present, it is the carboxy terminal part of the sequence and wherein Val 24 may be preceded by Gly; Z is absent or is all or a part of the sequence Pro 110 - Lys 156 shown in Figure 7, with the proviso that if part of the sequence is present, it is the amino terminal part of the sequence. Also provided by the present invention is a method of producing the polypeptide in bacteria, and a method of recovering the polypeptide produced in bacteria. In additional aspects, the present invention provides uses of the polypeptide in therapeutic and diagnostic methods applied in situations or conditions characterized by the occurrence of excessive blood coagulation and thrombosis. In an additional aspect, the present invention provides an antibody to the polypeptide. Brief Description of the Figures Figure 1: Purification and Identification of FXa Inhibitor In Leech Saliva This figure summarizes two different protocols used to purify a FXa inhibitor from DLS for amino acid sequencing, as well as various experiments performed in order to corroborate the identity and homogeneity of the resulting preparations. In Run II, Mono-Q was used instead of Q-Sepharose. Figure 2: Construction of Plasmid pλS0D-NGE-XaI-13"m" The construction of plasmid pλSOD-NGE-XaI-13"m" as described in Example 10 is shown. Plasmid pλS0D-NGE-XaI-13"m" expresses a fusion protein containing 63 amino acids of a modified Cu/Zn-SOD sequence and the FXal 13"m"polypeptide having the sequence glu^-lys 156 shown in Figure 7 under control of the λP L promoter. The 13"m" polypeptide may be obtained by hydroxylamine cleavage at the engineered cleavage site between the SOD moiety and the FXal moiety. Plasmid pλSOD-NGE-XaI-13 n m" has been deposited in the ATCC in E. coli 4300 under Accession No. 69269. Figure 3: Amino Acid Sequence Comparison of FXa Inhibitor Isolates and Antistasin This figure compares the amino acid sequences of naturally occurring FXa inhibitor as represented by clone PCR4, cDNA clones and antistasin. The sequences were aligned by the alignment program Pileup, based on: "Simplification of Progressive Alignment Method", Feng & ■ Doolittle, J. Mol. Evol. 3_5:351-360 (1987). PCR4 is the sequence of the PCR derived DNA sequence encoding a FXa inhibitor present in DLS and represents the sequence of the naturally occurring FXa inhibitor. (Methionine in the fourth position is apparently an error of the PCR reaction which explains why it differs from isoleucine in the fourth position of the N-terminal amino acid sequence obtained for the naturally occurring inhibitor isolated from leech saliva) . PCR4 was obtained as described in Example 3 by hybridization of PCR-derived clones with the nucleotide sequence encoding the first 9 amino acids of naturally occurring FXa inhibitor isolated from leech saliva. 13 represents the sequence of clone 13 from a cDNA library, and includes a putative extension peptide (see Example 4) . AS represents the sequence of antistasin. "<E" represents pyroglutamate. The numbering is according to the sequence of PCR4. Gaps have been introduced to obtain the best alignment. The cysteine residues which have been aligned are highlighted. Figure 4: Cloning of FXa Inhibitor cDNA by PCR As described in Example 3, poly A + mRNA was isolated from total RNA extracted from 120 leeches. An aliquot of the poly ' ' mRNA so obtained (5μg) was used as template in a reverse transcription reaction in the presence of the synthetic primer A A: 5'- AACTCGAGGATCCAAGCTTTTTTTTTTTTTTT - 3' and 4 dNTPs. Following the synthesis of single stranded complementary DNA (sε-cDNA) , the mRNA was degraded by overnight alkali treatment comprising 0.3M NaOH at room temperature. The neutralized ss-cDNA was subjected to PCR a plification using Taq polymerase, 4 dNTPs and the synthetic degenerative DNA oligomer B T A T T T B: 5' - CCGAATTCATATGTA GA GTIATITA GTIGA GA CC - 3' C G C C C as reverse primer. The PCR amplification products were digested with EcoRI and Hindlll. The gel purified fragments were then subcloned into the large EcoRI-Hindlll fragment of plasmid pSP65. The ligation mixture was used to transform E. coli MC1061. The resulting transformants were screened by in-situ hybridization using the radiolabeled synthetic probe C (disclosed in Example 3) corresponding to N-terminal amino acids 14 to 19 of the purified leech derived FXa inhibitor. Figure 5: DNA and Deduced Amino Acid Sequence of Clone DSP65-XaI-4 Plasmid DNA was prepared from positive clones identified by in-situ hybridization with the radiolabeled probe C. The purified plasmid DNA of clone pSP65-XaI-4 was sequenced by the Sanger dideoxy method. The resulting sequence was processed using LKB 2020 DNASIS software system. Figure 6: Cloning of FXa Inhibitor cDNA from a cDNA Library As described in Example 4, poly A* mRNA obtained from total RNA extracted from 120 leeches was used for double stranded complementary DNA (ds-cDNA) synthesis using the Stratagene ZAP™ cDNA synthesis kit. The ds-cDNA so obtained was digested with Xhol and EcoRI and subcloned into the Xhol- EcoRI digested Uni-ZAP vector designated pBluescript SK. The cDNA library obtained was screened for clones encoding FXa inhibitor, using radiolabeled DNA of plasmid pSP65-XaI-4 as a probe, under hybridization conditions of high and low stringency. The prehybridization of the filters was carried out for 8 hours at 60°C in 6x SSC (lx SSC: 0.15M NaCl, 0.015M Na-citrate) , 0.1% SDS, 5X Denhardt (0.1% Ficoli 400, 0.1% polyvinyl pyrrolidone, 0.1% ' BSA, 0.5% SDS) and lOOμg/ml salmon sperm DNA, followed by hybridization with the radioactive probe for 48 hours at 60°C. Following hybridization, the filters were washed at two different conditions: to obtain high stringency conditions, filters were washed with 0.2-0.5 x SSC containing 0.2% SDS at 65°C for two hours with several changes of solution; to obtain low stringency conditions, filters were washed with 2x SSC containing 0.2% SDS at 60°C for two hours with several changes of solution. The plasmid of one of the clones, obtained at low stringency, was designated pSK-XaI-13. Figure 7: DNA and Deduced Amino Acid Sequence of cDNA Clone DSP65-XaI-13 The construction of plasmid pSP65-XaI-13 containing DNA encoding FXal obtained from plasmid pSK-XaI-13 is described in Example 6. Purified DNA of plasmid pSP65-XaI-13 was sequenced by the Sanger dideoxy method. The data obtained was processed using the LKB 202C DNASIS software system. Figure 8: Subcloning of cDNA encoding recombinant FXal This figure shows the construction of plasmid pSP65-XaI-13 containing DNA encoding FXal obtained from plasmid pSK-Xal- 13 as described in Example 6. Plasmid pSK-XaI-13 was digested with Xhol and EcoRI. The Xhol-EcoRI fragment containing the FXal coding region was isolated and subcloned into Sail-EcoRI digested plasmid pSP65. The resulting plasmid was designated pSP65-XaI-13. Figure 9: Construction of Plasmid pSP65-XaI-13"m" Plasmid pSP65-XaI-l3 does not contain an ATG initiation codon at the predicted N-terminus of the mature protein. This figure shows the introduction into plasmid pSP65-XaI-13 of an ATG initiation codon adjacent to amino acid Val 24 by site-directed mutagenesis. Plasmid pSP65-XaI-13 was digested with EcoRI and Clal which cleaves within the FXal coding region. A second aliquot of plasmid pSP65-XaI-13 was digested with X nl and Seal which cleaves within the Amp R coding region. From both aliquots, the large DNA fragment was isolated, denatured and annealed in the presence of synthetic oligomer D: Ndel D: 5' - GAATTGCGAAGCATATGGTAATAGCAG - 3' thus introducing the initiation codon ATG . A DNA poly erase (Klenow) reaction was then carried out in the presence of 4 dNTPs followed by ligation with T 4 DNA ligase. A clone both ampicillin resistant and positive in hybridization with oligomer D was designated pSP65-XaI-13"m" . Figure 10: Construction of Expression Plasmid pλ-XaI-13"m" This figure shows the construction of a plasmid for the expression of a mature ("m") FXal polypeptide encoded by clone pSP65-XaI-13"m n under control of the λP L promoter. Plasmid pSP65-XaI-13 n m" was digested with Ndel and Hindlll. The Ndel-Hindlll DNA fragment coding for the FXal polypeptide was isolated and ligated to the large fragment isolated following Ndel-Hindlll digestion of plasmid pMAL- 183/191 containing the λP L promoter and the λcll ribosomal binding site (RBS) . The ligation mixture was used to transform E. coli 4300. The resulting plasmid which expresses FXal was designated pλ-XaI-13"m" and deposited under ATCC Accession No. 69135. Figure 11: Construction of Plasmid pDeo-S-XaI-13"f" for Expression of an SOD-FXal Fusion Protein This figure shows the construction of a plasmid under control of the deo P j P 2 promoters for expression of the recombinant FXal polypeptide produced by clone 13, fused to a polypeptide fragment containing 58 amino acids from a modified Cu/Zn-SOD sequence. Plasmid pSP65-XaI-13 was digested with Sspl and Pstl. The SspI-PstI DNA fragment encoding FXal was isolated and ligated to the large Scal- Pstl fragment of plasmid pBAST-R. The resulting plasmid was designated pDeo-S-XaI-13"f" and deposited in the ATCC under Accession No. 69137. Figure 12: Construction of Plasmid pλ-XaI-13"l" for Expression of an FXal Prepeptide (FXal sequence preceded by an extension peptide) This figure shows the construction of a plasmid under control of λPL for expression of a "prepeptide" of FXal. Plasmid pSP65-XaI-13 was modified by introduction of an Ndel site at met 3 by site-directed mutagenesis utilizing the PCR technique. DNA of plasmid pSP65-XaI-13 was PCR amplified in the presence of the 5' synthetic primer: I: 5' - CCGAATTCATATGTGTTGGAACAAAGGT - 3' and the synthetic oligomer: J: 5' - CCAAGCTTGGGCTGCAGGTCGA - 3' The product was digested with Ndel and Hindlll. The small Ndel-Hindlll fragment was then ligated to the large Ndel- Hindlll fragment of plasmid pMAL-183/191 containing the λP L promoter and the λcll RBS. The resulting plasmid was designated pλ-XaI-13"l" . Figure 13: Construction of Plasmid pDSOD-NGE-Xal-13" " The construction of plasmid pDSOD-NGE-XaI-13"m" as described in Example 10 is shown. Plasmid pDSOD-NGE-XaI-13"m" expresses a fusion protein containing 63 amino acids of a modified Cu/Zn-SOD sequence and the FXal 13"m"polypeptide having the sequence glu^-lys 156 shown in Figure 7 under control of the deo promoter. The 13"m" polypeptide may be obtained by hydroxylamine cleavage at the engineered cleavage site between the SOD moiety and the FXal moiety. Oligomer K and oligomer L have the following sequences: AsnGly Oligomer K: 5' - GTCCTAATGGTGAAGTAATAGCAGAGAAT - 3' Oligomer L: 3' - GATTACCACTTCAITATCGTCTCTTA - 5' Figure 14: Construction of expression plasmid pλ-XaI-13"m"- 05M Plasmid pλ-Xal-13"m" (ATCC Accession No. 69135) was digested with Ndel endonuclease. The linear form, large DNA fragment was isolated and ligated in the presence of T 4 DNA ligase to a synthetic DNA fragment having the following sequence: Gly 5' - TATG'GGT' GTA'ATA'GCA'GAGAAT -3' 3' - AC CCA CAT TAT CGT CTCTTA -5' After overnight ligation at 14°C, the ligation mixture was digested with Aatll endonuclease and the large DNA fragment was isolated. This DNA fragment was ligated to a Aatll-Sspl small fragment derived from another aliquot of plasmid pλ- Xal-13"m" previously digested with Aatll and Sspl endonucleases. The newly obtained plasmid was designated pλ-XaI-13"m"-05M. Figure 15: Construction of expression plasmid pλ-Xal-13"m"- Plasmid pλ-XaI-13"m"-05M was digested with Hindlll endonuclease and the linear DNA was isolated and ligated to the small Hindlll-Hindlll T,T 2 DNA fragment obtained by Hindlll endonuclease digestion of DNA plasmid pHG44 (deposited under ATCC Accession No. 39806 on August 20, 1984) . The newly obtained plasmid was designated pλ-Xal- 13"m"-05M-T,T 2 . Figure 16: Pharmacokinetics of FXal in the rabbit FXal was radio-iodinated ( s I) , admixed with unlabeled material (specific activity 0.13 x IO 6 cpm/μg) and injected in a rabbit (10 μg/rabbit) . Analysis of plasma samples at the indicated time intervals over a two hour period revealed that the labeled material was fully TCA precipitable at all times and its level -declined with a half-life of about 94 minutes. Figure 17: Pharmacodynamics of FXal in the rabbit Ex vivo APTT dose responses were measured at the indicated time intervals over a two hour period and revealed that in the rabbit the half-life value of FXal in the plasma was about 70-78 minutes. Figure 18: Three-injection regimen of FXal for the Rat Coil Model In order to attain a continuous plasma level of FXal, plasma levels of FXal were estimated by ex vivo APTT measurements which revealed that satisfactory plasma profiles (about 20% fluctuations) of FXal were obtained by an initial i.v. dose at zero-time, followed by two doses of 35% of the initial dose at 45 min intervals. Figure 19: The effect of FXal on clot formation in the rat coil model The weight of the clot formed on the steel coils was determined in the rat coil model (as described in Example 15) with three doses of FXal as indicated. Figure 20: Effect of FXal on tail bleeding in mice Tail bleeding was determined in mice injected i.v. with various doses of FXal as indicated, five minutes after injection of the FXal. Figure 21: Effect of heparin on tail bleeding in mice Tail bleeding was determined in mice injected i.v. with various doses of heparin as indicated, five minutes after injection of the heparin. Figure 22: The effect of FXal on acute arterial thrombosis in the rat The decrease in arterial temperature (%) was determined in an acute arterial thrombosis model in the rat (as described in Example 17) after correction for changes in body temperature. For the two higher doses, the difference from the control is statistically significant (p<0.01-0.05 at 20 and 40 min and p<0.001-0.01 at 60 min) , whereas at an FXal dose of 0.133 mg/kg, the difference is not statistically significant. Figure 23: Inhibition of arterial thrombosis in the baboon bv FXal Three different baboons were each injected with three distinct doses of FXal as indicated. One baboon was injected with PBS and served as a control. *** In-platelet deposition was measured on the dacron graft at the indicated time intervals by a gamma camera. Figure 24: Inhibition of venous thrombosis in the baboon by FXal Three different baboons were each injected with three distinct doses of FXal as indicated. One baboon was injected with PBS and served as a control. *II In-platelet deposition was measured in the chamber at the indicated time intervals by a gamma camera. Figure 25: Arterio-venous shunt system A schematic drawing of the thrombogenic device used in Example 18 is depicted. Figure 26: Construction of expression plasmid pλ-Xal-13 "m" - T,T 2 Plasmid pλ-Xal- 13 "m" (Figure 10) was digested with Hindlll endonuclease and the linear DNA was isolated and ligated to the small Hindlll-Hindlll T,T 2 DNA fragment obtained by Hindlll endonuclease digestion of DNA plasmid pHG44 (deposited under ATCC Accession No. 39806 on August 20, 1984) . The newly obtained plasmid was designated pλ-Xal- 13"m π -T,T 2 . Detailed Description of the Invention Abbreviations The following abbreviations are used as amino acid designations: A Ala Alanine C Cys Cysteine D Asp Aspartic acid E Glu Glutamic acid F Phe Phenylaline G Gly Glycine H His Histidine I He Isoleucine K Lys Lysine L Leu Leucine M Met Methionine N Asn Asparagine P Pro Proline 0 Gin Glutamine R Arg Arginine S Ser Serine T Thr Threonine V Val Valine W Trp Tryptophan Y Tyr Tyrosine The present patent application discloses the identification and characterization of a novel and heretofore unknown polypeptide derived from the leech Hirudo medicinalis. This recombinant polypeptide is an inhibitor of FXa and is herein designated "FXal". Recombinant FXal is also termed Yagin. In another aspect, the present application discloses the complete gene sequence as well as the deduced amino acid sequence of the novel polypeptide. In yet another aspect, the present application discloses production in bacteria of the novel polypeptide, and its refolding to yield a biologically active protein having inhibitory activity towards the enzymatic activity of FXa. The invention provides a polypeptide (protein) comprising the amino acid sequence X - Y - CYS GLN GLU GLU GLU CYS PRO ASP PRO TYR LEU CYS SER PRO VAL THR ASN ARG CYS GLU CYS THR PRO VAL LEU CYS ARG MET TYR CYS LYS PHE TRP ALA LYS ASP GLU LYS GLY CYS GLU ILE CYS LYS CYS GLU GLU LEU CYS GLN ASN GLN ASN CYS THR LYS ASP MET LEU CYS SER SER VAL THR ASN ARG CYS ASP CYS GLN ASP PHE LYS CYS PRO GLN SER TYR CYS - Z wherein X is MET or absent; Y is 0-29 amino acids of the sequence LYS MET CYS TRP ASN LYS GLY CYS PRO CYS GLY GLN ARG CYS ASN LEU HIS ARG ASN GLU CYS GLU VAL ILE ALA GLU ASN ILE GLU, with the proviso that if part of the sequence is present, it is the carboxy-terminal part of the sequence and wherein Val 24 may be preceded by Gly; and Z is absent or all or a part of the sequence Pro 110 - Lys 15t> shown in Figure 7, with the proviso that if part of the sequence is present, it is the amino-terminal part of the sequence. The polypeptide may be glycosylated or non-glycosylated. It is envisaged that the recombinant polypeptide of the subject invention encompasses polypeptides which are homologs of the above described polypeptide. In preferred embodiments, the polypeptide has the amino acid sequence A-Lys 2 -Lys li6 , Ile 29 -Lys 156 , Glu^-Lys 156 , or A-Val^-Lys 156 where Lys -Lys 156 , Ile 29 -Lys 156 , Glu^-Lys 156 , and Val^-Lys 156 are identical to the sequence shown in Figure 7, and wherein A is MET or absent and wherein Val 24 may be preceded by Gly. In an additional aspect, the subject invention provides for biologically active recombinant FXal. Biologically active FXal is defined as FXal which has inhibitory activity towards the enzymatic activity of Factor Xa as measured by the biochemical activity assay described in Example 2. Inhibitory activity is defined as activity which reduces the enzymatic activity of Factor Xa as measured by the above mentioned biochemical assay. As a result of the lowering of the enzymatic activity of FXa, the extent of blood coagulation is reduced. It is contemplated that the polypeptide of the invention may have alternative or additional biological activity or activities instead of or in addition to being an inhibitor of FXa; in particular, it may have inhibitory activity to other factors in the coagulation cascade. As used herein, a homolog of the polypeptide of the invention is a polypeptide which has substantially the same amino acid sequence and biological activity as the polypeptide. Thus, a homolog may differ from the polypeptide of the invention by the addition, deletion, or substitution of one or more non-essential amino acid residues, provided that the resulting polypeptide retains the biological activity of the polypeptide. Persons skilled in the art can readily determine which amino acids residues may be added, deleted, or substituted (including with which amino acids such substitutions may be made) using established and well known procedures, including, for example, conventional methods for the design and manufacture of DNA sequences coding for bacterial expression of polypeptide homologs of the subject polypeptide, the modification of cDNA and geno ic sequences by site-directed mutagenesis techniques, the construction of recombinant proteins and expression vectors, the bacterial expression of the polypeptides, and the measurement of the biochemical activity of the polypeptides by means of conventional biochemical assays. Examples of homologs are deletion homologs containing less than all the residues specified in the subject polypeptide, substitution homologs wherein one or more residues specified are replaced by other residues, and addition homologs wherein one or more amino acids residues are added to the polypeptide. All such homologs share the biological activity of the polypeptide of the invention. Substantially the same amino acid sequence is herein defined as encompassing the addition or deletion of fewer than four amino acids at the N-terminus of the amino acid sequence of FXal. Furthermore, there may be substitutions and/or deletions in the sequence which do not eliminate the biological activity of the protein. Such substitutions are known to those skilled in the art. Substitutions may encompass up to 10 residues in accordance with the homologous or equivalent groups described by e.g. Lehninger, Biochemistry. 2nd ed. Worth Pub., N.Y. (1975); Creighton, Protein Structure, a Practical Approach, IRL Press at Oxford Univ. Press, Oxford, England (1989); and Dayhoff, Atlas of Protein Sequence and Structure 1972. National Biomedical Research Foundation, Maryland (1972) . Substantially the same biological activity refers to biological activity the same as that of the polypeptide possibly differing slightly in degree or level which would still be known by the skilled artisan to be the same biological activity. The present application discloses DNA isolated from an mRNA derived cDNA library of Hirudo medicinalis. The polypeptides encoded by this DNA or a part thereof are inhibitors of FXa, but the amino acid sequence deduced from the DNA nucleotide sequence is substantially different from the sequence of the known naturally occurring FXa inhibitor disclosed by Rigbi (13) . There is no prior disclosure of the existence of this novel polypeptide. The polypeptide of the invention may be obtained as a "mature" protein, i.e. in the absence of any extension or fusion peptides, as prepeptides containing leader or extension peptides, or as fusion peptides containing all or a portion of another protein or polypeptide. Mature polypeptide may be obtained by direct expression or by cleavage of a fusion polypeptide or prepeptide. In a specific embodiment, the recombinant FXal is expressed as a mature polypeptide (i.e. lacking any amino terminal or carboxy terminal extensions such as leader peptides or fusion proteins) by E.coli 4300 containing plasmid pλ-Xal- 13"m" or plasmid pλ-XaI-13 n m"-T,T 2 or plasmid pλ-XaI-13"m"- 05M-T-T 2 under control of the λP L promoter. In another embodiment, the recombinant FXal is produced as a fusion polypeptide by E.coli 733 containing plasmid pDeo- S-XaI-13"f" under control of the deo promoter which expresses recombinant FXal fused to the first 58 N-terminal amino acids of a modified Cu/Zn-SOD sequence. Cu/Zn-SOD is described in coassigned U.S. Patent No. 4,742,004 and by Steinman, H.M. , Superoxide Dismutase. (Oberley, ed.) CRC Press, Florida, pages 11-68, 1982.) In yet another embodiment, the recombinant FXal is obtained as a prepeptide containing a putative extension sequence expressed by E.coli 4300 containing plasmid pλ-XaI-13"1" . In a more preferred embodiment, the recombinant FXal is produced as a fusion polypeptide by plasmid pλSOD-NGE-Xal- 13"m" which encodes a fusion polypeptide containing a 63 amino acid fragment of a modified Cu/Zn-SOD sequence, a hydroxylamine cleavage site, and the FXal 13"m" polypeptide having the sequence glu^-lys 156 shown in Figure 7. One skilled in the art may readily obtain the FXal 13"m" polypeptide by cleavage of the fusion protein at the engineered cleavage site. Mature recombinant FXal may thus be obtained by direct expression or by cleavage of a fusion protein or prepeptide by known methods. Furthermore, one skilled in the art knows how to obtain the mature recombinant FXal from other fusion peptides and other manipulations of the DNA encoding FXal DNA using known and readily available materials and methods (e.g. Nilsson et al. , Current Opinion in Structural Biology, 2:569-575 (1992) and Hopp et al. ' , BioTechnology :1204-1210 (1988) ) . The subject invention further provides DNA encoding the polypeptides of the subject invention. The subject invention also provides expression plasmids comprising the DNA encoding the polypeptides of the subject invention. Examples of vectors that may be used to express DNA encoding the polypeptides are viruses such as bacterial viruses, e.g., bacteriophages (such as phage lambda), cosmids, plasmids, and other vectors. Genes encoding the relevant polypeptides are inserted into appropriate vectors by methods well known in the art. For example, using conventional restriction endonucleases, inserts and vector DNA can both be cleaved to create complementary ends which pair with each other and are then ligated together with a DNA ligase. Alternatively, -synthetic linkers harboring base sequences complementary to a restriction site in the vector DNA can be ligated to the insert DNA, which is then digested with the restriction enzyme which cuts at that site. Other means are also available and are known to those skilled in the art. Vectors comprising a sequence encoding the polypeptide " may be adapted for expression in a range of prokaryotic and eukaryotic host cells e.g. bacteria, fungi, yeast, plant, insect, and mammalian cells such as CHO, chicken embryo, fibroblast, kidney and other known cell lines. These vectors additionally comprise regulatory elements necessary for expression of the cloned gene located relative to the sequence encoding the polypeptide so as to effect expression of the polypeptide in the host. Examples of regulatory • elements required for expression include promoters, operators, and ribosomal binding sites. For example, a bacterial expression vector may include a promoter-operator sequence such as λ P L 0 L or deo promoters and operators. For initiation of translation, ribosomal binding sites such as λC π or deo ribosomal binding sites may be used. Additional appropriate elements such as repressors and enhancers may also be present. Those skilled in the art know how to use regulatory elements appropriate for various expression systems. The regulatory elements are positioned within the plasmid relative to the DNA encoding the polypeptides so as to effect expression of the polypeptides in. a suitable host cell. In preferred embodiments of the invention, the regulatory elements are positioned close to and upstream of the DNA encoding the polypeptides. Other suitable regulatory elements are known to those skilled in the art. Such vectors may be obtained commercially or constructed from the sequences described by methods well known in the art, for example U.S. Patent No. 4,831,120, issued May 16, 1989 and U.S. patent No. 5,143,836, issued September 1, 1992, which disclose methods concerning λP L , and European Patent Application Publication No. 303,972, published February 22, 1989 which discloses methods concerning the deo promoter. The invention provides plasmids encoding any of the recombinant proteins described above. A preferred embodiment is plasmid pSP65-XaI-13, deposited under ATCC Accession No. 69134 on December 1, 1992. Plasmid pSP65-XaI-13 does not express any protein since it lacks suitable regulatory elements; however, one skilled in the art knows how to manipulate the DNA of plasmid pSP65-XaI-13 in order to obtain expression of recombinant FXal. In more preferred embodiments, these plasmids are expression plasmids designated pDeo-S-XaI-13"f" deposited under ATCC No. 69137 on December 1, 1992, plasmid pλ-XaI-13"m" deposited under ATCC Accession No. 69135 on December l, 1992, and plasmid pλSOD-NGE-XaI-13"m" deposited under ATCC Accession No. 69269 on March 23, 1993. In additional preferred embodiments, the expression plasmids are plasmids pλ-XaI-13"m"-T,T 2 , pλ-XaI-13"m"-OSM-TiT-*, and pλ- XaI-13"m"-05M. Those skilled in the art will understand that the plasmids deposited in connection with this application may be readily altered by known techniques (e.g. site-directed mutagenesis or insertion of linkers) to encode expression of related polypeptides (homologs) . Such techniques are described for example in Sambrook, J., Fritsch, E.F. and Maniatis, T. (1989) Molecular Cloning: A Laboratory Manual, 2nd edition, Cold Spring Harbor Laboratory Press. The expression plasmids of the invention may be introduced into suitable host cells, preferably bacterial host cells. However, those skilled in the art will understand that the expression plasmids of this invention may be suitably modified for introduction into a range of prokaryotic and eukaryotic host cells as described above. The bacteria used as hosts may be any strains including auxotrophic (such as E. coli A1645) , prototrophic (such as E. coli A4255) , and lytic strains; F" " and F " strains; strains harboring the cl 857 repressor sequence of the λ prophage such as E. coli A1645 and A4255) ; and strains devoid of the deo repressors and/or the deo gene (see European Patent Application Publication No. 0303972, published February 22, 1989) . Escherichia coli A4255 has been deposited under ATCC Accession No. 53468, and Escherichia coli Sφ930 has been deposited under ATCC Accession No. 67706. In preferred embodiments, E. coli 4300 is used as host for expression plasmids under control of λ P L and E. coli 733 is used as host for expression plasmids under control of the deo promoter. Preferred bacterial host cells are Escherichia coli cells. Examples of suitable Escherichia coli cells are strains 733 and 4300 or 4300 (F) , but other Escherichia coli strains and other bacteria can also be used as hosts for the plasmids. An example of a eukaryotic cell is an insect cell. Preferred insect cells are Sf-9 cells harboring a baculovirus expression system. The invention provides a host-plasmid system which comprises a bacterial cell with any of the aforementioned expression plasmids. All the E. coli host strains described above can be "cured" of the plasmids they harbor by methods well-known in the art, e.g. the ethidium bromide method described by R.P. Novick (1969) in Bacteriol . Review 33. 210. One skilled in the art knows how to produce the polypeptides disclosed herein from different plasmids and/or different nucleotide sequences encoding the same amino acid sequences due to the degeneracy of the genetic code. One skilled in the art can also produce substantially identical homologs having small changes in the amino acid sequence which do not affect the structure or specific biological activity of the polypeptide. Such homologs are also encompassed by the invention. In addition, the invention provides a method of producing biologically active recombinant FXal which comprises transforming a host cell with an expression plasmid encoding the polypeptide, culturing the transformed host cell so that the cell produces the polypeptide encoded by the plasmid, and recovering the polypeptide so produced. In another aspect, the invention provides a method for producing a biologically active recombinant FXal polypeptide wherein the host cell is a bacterial cell, and the recovering comprises: (a) disrupting the cells so as to produce a lysate containing the polypeptide; (b) treating the lysate so as to obtain inclusion bodies containing the polypeptide; (c) treating the inclusion bodies so as to obtain the polypeptide in soluble form; (d) treating the resulting soluble polypeptide so as to form biologically active polypeptide; (e) recovering the biologically active polypeptide so formed; and (f) purifying the biologically active polypeptide so recovered. In a preferred embodiment, the treating of step (c) comprises the addition of a denaturant, the treating of step (d) comprises contacting the polypeptide with a mixture of a thiol-containing compound and a disulfide, and the purifying of step (f) comprises column chromatography. In a more preferred embodiment, the denaturant is guanidinium chloride or urea, the thiol containing compound is glutathione, thioredoxin, β-mercaptoethanol, or cysteine, the disulfide is oxidized glutathione, cystine, or the product of air oxidation of mercaptoethanol, and the column chromatography comprises either one or both of Q-Sepharose chromatography and Heparin-Sepharose chromatography or either one or both of Q-Sepharose chromatography and S- Sepharose chromatography. The invention further provides a composition comprising any of the polypeptides described effective to obtain a desired therapeutic effect resulting from the biological activity of the polypeptide, and a suitable carrier. In a preferred embodiment, the desired therapeutic effect is the reduction of the extent of blood coagulation and thrombosis. The extent of blood coagulation may be represented by in vitro coagulation assays such as APTT. The invention also provides a method of reducing the extent of blood coagulation comprising contacting the blood with an amount of the claimed polypeptide effective to reduce the extent of blood coagulation. In a preferred embodiment, the contacting is effected in vivo in a subject. In a more preferred embodiment, the subject suffers from excessive blood coagulation. In particular embodiments, the subject suffering from excessive blood coagulation has a condition selected from the group consisting of vascular disorders, post-operative trauma, tendency towards venous thromboembolism associated with obesity, pregnancy, the use of oral contraceptives, and prolonged immobilization. Accordingly, -the invention provides a method of reducing the extent of blood coagulation and thrombosis in a cerebrovascular disorder such as stroke or other cerebrovascular disorders. The invention provides a method of reducing the extent of blood coagulation in a condition of excessive blood coagulation such as occurs in thrombosis, more particularly venous thrombosis and more particularly deep venous thrombosis or disseminated intravascular coagulation. The invention further provides a method of reducing the extent of blood coagulation in a condition of excessive blood coagulation such as occurs in arterial thrombosis such as thrombosis of a coronary artery. As described above, thrombosis often occurs following thrombolysis. Accordingly, the invention provides a method of reducing the extent of blood coagulation following thrombolysis. In a preferred embodiment, the thrombolysis is effected with a fibrinolytic agent. In particular embodiments, the fibrinolytic agent is tissue plasminogen activator or streptokinase. The polypeptide may be administered before, during, or after the administration of the fibrinolytic agent, or bound to the fibrinolytic agent. The invention also provides a method of inhibiting the activity of Factor Xa which comprises contacting Factor Xa with an amount of the polypeptide effective to inhibit the activity of the Factor Xa. In an additional embodiment, the polypeptide of the invention may be used to prevent recurrent influenza infection. Influenza infection is a dynamic process comprising infection and reinfection of cells by the virus. The activation enzyme implicated in A-type influenza • infection has been shown to be very similar to chicken blood coagulation Factor Xa (Gotoh B. et al. (1990), EMBO J. 9_: 4185-4190 and Ogasawara T. et al (1992), EMBO J. 11: 467- 472) . It is therefore possible that the corresponding human FXa is involved in influenza infections occurring in humans. It is envisioned that an FXa inhibitor would be useful in preventing recurrent influenza infection. In a specific embodiment, the FXa inhibitor would be .the polypeptide of the invention. It is also contemplated that the polypeptide might be administered in conjunction with additional therapeutic agents. In a particular embodiment, the additional therapeutic agent comprises an oxygen free radical scavenger. In a preferred embodiment, the oxygen free radical scavenger is superoxide dismutase. In still more preferred embodiments, the superoxide dismutase is Cu/Zn-SOD (U.S. Patent No. 4,742,004) or MnSOD (coassigned U.S. Serial No. 842,740, filed February 27, 1992 and UK Patent No. GB 2,183,658,* April, 25, 1990). Such treatment would have numerous advantages over presently available therapeutic and prophylactic methods of handling influenza infection. Presently used methods of influenza prevention are based on immunization against the influenza virus. This method has proved unreliable due to the high frequency of mutation of the influenza virus, as well as the large number of viral strains. Use of an inhibitor of the mechanism of infection would therefore be greatly advantageous by not being based on the immunological properties of the viral particle, and therefore not limited to a particular strain. Furthermore, since influenza is a bronchopulmonary disease, the treatment would preferably be administered as an aerosol, thus providing a simple method of administration. In an additional aspect, the invention provides an antibody which specifically reacts with an epitope of the polypeptide. In a particular embodiment, the antibody is a monoclonal antibody. The invention also encompasses a polypeptide which competitively inhibits the specific reacting of the antibody. Plasmids and Strains Deposited in Connection with the Present Invention: Various plasmids and E. coli strains useful in carrying out the methods of the invention were deposited with the American Type Culture Collection, 12301 Parklawn Drive, Rockville, Maryland 20852 pursuant to and in satisfaction of, the requirements of the Budapest Treaty on the International Recognition of the Deposit of Microorganisms for the Purposes of Patent Procedure. These plasmids include plasmid pSP65-XaI-13 deposited in EL. coli MC1061 under ATCC Accession No. 69134 which contains cDNA encoding FXal of clone 13; plasmid pDeo-S-XaI-13"f" deposited in E. coli 733 under ATCC Accession No. 69137 which expresses FXal protein under control of the deo promoter fused to 58 N-terminal amino acids of a modified sequence of human Cu/Zn-SOD; pλ-XaI-13"m" deposited in E. coli 4300 under ATCC Accession No. 69135 which expresses a mature FXal polypeptide. These aforementioned plasmids were deposited on December 1, 1992. Plasmid pλS0D-NGE-XaI-13"m" which was deposited in E. coli 4300 (F " ) on March 24, 1993 under ATCC Accession No. 69269 expresses a cleavable fusion protein containing a 63 amino acid fragment of a modified Cu/Zn-SOD sequence, a hydroxylamine cleavage site, and the FXal 13"m" polypeptide. Deposited bacterial strains are E. coli 4300 which is a host cell for plasmids controlled by the λ P L promoter, deposited as noted above under ATCC Accession No. 69135 and E. coli 733 which is a host cell for plasmids controlled by the deo promoter, deposited as noted above under ATCC Accession No. 69137. Additionally, plasmid pMLK-100, a vector harboring the λP L promoter, was deposited in E. coli 4300 under ATCC Accession No. 68605 on February 6, 1991 and plasmid pHG44, a vector harboring the T1T2 transcription termination sequence was deposited in E. coli under ATCC Accession No. 39806 on August 20, 1984. EXAMPLES The Examples which follow are set forth to aid in understanding the invention but are not intended to, and should not be construed so as to, limit its scope in any way. The examples do not include detailed descriptions for conventional methods employed in the isolation of cDNA, the construction of vectors, the insertion of genes encoding polypeptides of interest into such vectors, or the introduction of the resulting plasmids into bacterial hosts. Such methods are well known to those skilled in the art and are described in numerous publications including Sambrook, Fritsch, and Maniatis, Molecular Cloning: A Laboratory Manual. 2nd Edition, Cold Spring Harbor Laboratory Press, USA, (1989) . Examples 1 and 2 describe the isolation and characterization of a naturally occurring FXa inhibitor from the saliva of the leech Hirudo medicinalis. Examples 3-18 describe how DNA encoding the polypeptide obtained above was used to identify a novel DNA sequence encoding the novel polypeptide of the present invention, and additional novel and inventive embodiments of the present invention. Example 1: Purification of Naturally Occurring FXa Inhibitor From Saliva of H. medicinalis An FXa inhibitor was purified from dilute leech saliva (DLS) of Hirudo medicinalis. DLS was obtained as described by Rigb et al. , Comp. Biochem. Physiol. 87B:567-573 (1987). In brief, a phagostimulatory solution of 0.001M arginine in saline kept at 37°C was placed over a washed membrane (sausage skin, obtained from Nippi Gelatin Casing, Tokyo, Japan) which was stretched across a cylinder open at both ends. Starved leeches were allowed to suck the solution into their huge crop until satiated. As the feeding solution was not stirred, it is assumed that most of the saliva excreted into it was reimbibed. Following cessation of feeding, the ingested solution containing the saliva was forced out through the mouth by squeezing the leech from the posterior end forwards. Colorless fluid, designated dilute leech saliva (DLS) , was collected. Two methods of purification of the proteins from DLS are described below. The two methods are similar and consist of anion exchange chromatography on a quaternary aminomethyl column (Mono-Q or Q-Sepharose) followed by either gel filtration chromatography on Superose 12 or by affinity chromatography on Heparin-Sepharose. In both cases, the identity and the homogeneity of the preparation were verified by SDS-PAGE and at least one supplementary method. These purification protocols are summarized in Figure 1. 1. Purification on Mono-0 followed by Superose 12 DLS obtained from about 25 leeches (150 ml) was diluted with an equal volume of 20mM Tris-HCl pH 8.9, and applied to an FPLC Mono-Q column (5x50 mm) . The column was eluted with a linear gradient of 0-0.5M NaCl in the same buffer (80 ml) at a flow rate of 1 ml/min. Absorbance was monitored at 280nm. The column was then washed with a second linear gradient of 0.5M-1M NaCl in the same buffer (20 ml) and at the same flow rate followed by an additional 5ml of 1M NaCl. Fractions of 1ml were collected and assayed for both FXa inhibitor activity and hirudin activity. ' The peak of FXa inhibitor activity eluted at about 0.35 M NaCl and was separate from the peak of hirudin activity. The fractions containing FXa inhibitory activity were pooled and applied to a Superose 12 column 10 x 900mm (three 10 x 300mm columns in series) . Elution was carried out at room temperature with 20 mM Tris- HCl pH 8 containing 150mM NaCl at a flow rate of 0.3 ml/min. Absorbance was monitored at 280nm. The FXa inhibitor activity eluted under these conditions as a single peak with an apparent molecular weight of 4kD. In contrast, electrophoresis on Tricine SDS-PAGE of the fraction containing the purified FXa inhibitory activity revealed a single band of molecular weight ' of about 14 kD. As will be described in Example 2, this inconsistency in molecular weight determination may be due to nonspecific interactions of the Superose 12 with the FXa inhibitor leading to retardation of elution of the FXa inhibitor from the column. The purified polypeptide was also analyzed by reverse phase HPLC (RP-HPLC) as follows. The FXa inhibitor, purified on Superose 12, was dialyzed against water, lyophilized, redissolved in 1ml of 0.1% trifluoroacetic acid (TFA) , and loaded onto a 7μ RP-300 column (Aquapore, Brownlee Labs) attached to a Kontron Model 420/422S HPLC. Elution was carried out with a linear gradient of 0-100% acetonitrile in 0.01% TFA (60ml) at a flow rate of 1 ml/min. Absorbance was monitored at 220nm. A single peak with a retention time of 38.51 minutes was observed thus demonstrating that the FXa inhibitor was purified to homogeneity. 2. Purification on 0-Sepharose followed by Heparin- Sepharose. DLS obtained from about 50 leeches (320 ml) was diluted with an equal volume of 20mM Tris-HCl pH 8.9, and applied to a Q- Sepharose column (10x50 mm) . The column was eluted with a linear gradient of 0-0.5M NaCl in the same buffer (70 ml) at a flow rate of 1 ml/min; absorbance was monitored at 280nm. The column was then washed with IM NaCl in the same buffer at the same flow rate. Fractions of 1ml were collected and assayed for both FXa inhibitory activity and hirudin activity. This verified that the FXa inhibitor peak was indeed separated from the hirudin peak. The FXa inhibitor eluted in a single peak at about 0.35M NaCl, which was similar to the elution from Mono-Q described above. The fractions containing FXa inhibitory activity were pooled and dialyzed against 20mM Tris-HCl pH 8.9. An aliquot of 7ml was then applied to a Heparin-Sepharose column (10x50 mm) . Elution was carried out with a linear gradient of 0-0.5M NaCl in the same buffer (70ml) at a flow rate of 1 ml/min and absorbance monitored at 280nm. The column was then washed with IM NaCl in the same buffer and at the same flow rate. Fractions of lml were collected and assayed for FXa inhibitory activity. The purified FXa inhibitor eluted in a single peak at about 0.30M NaCl. By this protocol, the FXa inhibitor was purified approximately 600-fold with an overall yield of 14%. As will be described below, Tricine SDS-PAGE analysis of this preparation revealed a single band with a molecular weight of 14 kD. The homogeneity of the FXa inhibitor purified by this procedure was further verified by gel permeation chromatography under denaturing conditions. Example 2: Characterization of FXa Inhibitor Isolated from DLS of Hirudo medicinalis 1. Molecular Weight 1.1 Tricine SDS-PAGE Various preparations of the FXa inhibitor prepared as described in Example l were analyzed to evaluate both the degree of homogeneity and the molecular weight of the protein. The protocol used was a modified version of the method of Schagger et al. (Anal. Biochem. 166:368-379 (1987) ) . The band corresponding to the FXa inhibitor migrated to a position of about 14kD and in some cases as a broader band of about 12-l4kD. 1.2. Gel Permeation Chromatography on BioGel P-60 Molecular weight was also determined by gel permeation chromatography of a crude preparation of the FXa inhibitor on BioGel P-60. FXa inhibitory activity eluted at a retention time corresponding to a molecular weight of 14 kD. As mentioned above, the molecular weight of the FXa inhibitor as determined under denaturing conditions by electrophoresis on Tricine SDS-PAGE was also 14 kD, thus demonstrating that the FXa inhibitor is a 14kD monomer. 2. N-terminal sequence Samples of FXa inhibitor purified from leech saliva by the two procedures described in Example 1 were further resolved by Tricine SDS-PAGE and electroblotted onto a PVDF membrane. The membrane slice containing the FXa inhibitor was subjected to automatic sequencing (Applied Biosystems Microsequencer, Model 475A) for 20 cycles. The following sequence was obtained for samples purified by both of the above described methods: Y - E - V - I - Y - V - D - D - P - C'- E - D - S - D - Tyr-Glu-Val-Ile-Tyr-Val-Asp-Asp-Pro-Cys-Glu-Asp-Ser-Asp- C * - E - D - G - N - K Cys-Glu-Asp-Gly-Asn-Lys * Cysteines are not detected by this method of amino acid sequencing and were later deduced from the DNA sequence. The same N-terminal sequence was obtained following pyroglutamate aminopeptidase treatment showing that there is no FXa inhibitor isoform which is blocked by pyroglutamate at its N-terminus. 3. Biochemical activity assay The assay of FXa inhibitory activity is based on the inhibition of the FXa-mediated hydrolysis of the chromogenic substrate methoxycarbonyl - D-cyclohexylglycyl - glycyl - arginyl p-nitroanilide (CHG) as described below. FXa activity is observed as the increase in absorbance at 405 run (e M = 9920 M" l cm '- ) , due to the release of p-nitroaniline. The kinetic parameters for hydrolysis of this substrate by FXa are as follows: catalytic constant = 130 sec '1 and 1^=15 μM. The appropriate substrate concentration was determined from the molar absorbance which is 8210 at 342 nm. The reaction mixture contains 50mM Tris-HCl pH 8.2, 5mM CaCl 2 , 200mM NaCl, 0.1% PEG, 40μM substrate, and approximately 2nM FXa. The FXa concentration is determined more precisely from the initial (first 20 seconds) rate of CHG hydrolysis at 40 μM in the absence of inhibitor based on the kinetic parameters mentioned above. Inhibition of the increase in absorbance may be observed in the presence of the FXa inhibitory activity of an FXa inhibitor such as FXal of the present invention. The inhibitor concentration is the difference in absorbance between the reaction in the presence and absence of inhibitor multiplied by the FXa concentration. This determination assumes the formation of a 1:1 enzyme-inhibitor complex, and further assumes that the dissociation constant (K,) is low enough (i.e., tight binding) to enable essentially total binding of the inhibitor (FXa inhibitor) to the enzyme (FXa) . However, analysis of the saturation shape of the dose-response curve (the degree of inhibition versus inhibitor concentration) of the naturally occurring FXa inhibitor isolated from the saliva of the leech provides evidence that a finite and non- negligible dissociation of the inhibitor-enzyme complex occurs under these conditions. The degree of dissociation of the FXa-FXa inhibitor complex is characterized by the inhibition constant (K,) and may be determined from the inhibited velocity of the hydrolysis of the chromogenic substrate CHG. These results may be analyzed according to kinetic schemes of competitive inhibition for enzyme-inhibitor complexes, essentially as described in Dixon and Webb, Enzymes (3rd ed.), Academic Press, New York, (1979) . The inhibitor concentration determined by the above method may therefore be underestimated. It is therefore useful to define the FXa inhibitor concentration as the concentration required to obtain a constant degree of inhibition, usually 50%, and to define the amount causing 50% inhibition (IC 50 ) under these reaction conditions to be l milliunit (mu) or 1 pmol. The FXa inhibitor activity assay used to determine concentration during purification and characterization was performed in an Elisa Titertek ' Twin Reader, Type 380 (EF LAB) or in a Phillips (Model PU 8720) UV/Vis scanning spectrophotometer. The inhibitory activity of a FXa inhibitor may also be assayed by its effect on FXa in a prothrombinase complex. The phospholipid used was rabbit brain cephalin (Sigma) . The contents of one vial, suspended in 1 ml 0.15M NaCl were diluted 1:40 in the reaction mixture, i.e. 2.5μl/100μl. The concentrations of the other components were: FXa 250 pM, prothrombin 2.67 μM, FVa 4.2 nM and Ca ++ 1 mM in 20 mM Tris- HCl pH 7.4/150 mM NaCl/0.1% polyethylene glycol 6000. Following 10 minutes of preincubation at 37°C, the reaction was initiated by the addition of prothrombin. Aliquots were removed at various time intervals. The reaction was stopped by lOmM EDTA and aliquots were kept on ice until assayed. The inhibition of FXa by a FXa inhibitor (e.g. FXal of the present invention) was observed as the effect of FXal on the generation of thrombin from prothrombin at 37°C. The amount of thrombin generated was assayed using 80μg of the synthetic thrombin β-nitroanilide substrate S-2238 (KabiVitrum, Sweden) at 23°C. The results obtained for naturally occurring FXa inhibitor were K, of 72pM and IC J0 of 1 0pM. In some later experiments (e.g. Example 14), slight modifications were made to the above protocol of the prothrombinase complex assay: the assay mix contained 50 pM FXa and 1.35 μM prothrombin instead of 250 pM FXa and 2.67 μM prothrombin resulting in a Ki of 120pM. Example 3: Cloning of FXa Inhibitor cDNA by PCR The following procedure is shown schematically in Figure 4. Total RNA was extracted from 120 leeches, Hirudo medicinalis. From the total RNA, 35 μg of poly A + mRNA were isolated (using the Fast Track™ mRNA isolation kit, Invitrogen) . An aliquot (5 μg) of the poly A + mRNA so obtained was used as template in a reverse transcription reaction in the presence of the following synthetic primer: Xhol Hindlll A: 5'- AACTCGAGGATςCAAGC , i"ri"l"ri"I"l"l"i"l"i"i"ri l - 3' BamHI The 15-mer oligo-dT sequence provides complementarity to the poly A sequence of the various mRNAs. Following synthesis of the single stranded complementary DNA (ss-cDNA), the mRNA was degraded by alkali treatment (0.3M NaOH overnight at room temperature) . An aliquot of the neutralized ss-cDNA was then subjected to PCR amplification using as reverse primer the following synthetic degenerative DNA oligomer: Ndel T A T T T B: 5' - CCGAATTCATATGTA GA GTIATITA GTIGA GA CC - 3' ECORI C G C C C This synthetic primer was prepared in accordance with the first nine N-terminal amino acids of the naturally occurring FXa inhibitor (Example 2) and encodes the sequence: NH 2 -Tyr-Glu-Val-Ile-Tyr-Val-Asp-Asp-Pro- (COOH) . The PCR amplification conditions were as follows: 1. Primer A 0.2μg 2. Primer B 0.2μg 3. ss-cDNA 5μl (5% of total) 4. 5mM dNTP 4μl 5. 10X PCR buffer lOμl 6. Taq polymerase 0.2μl (8U/μl (USB)) 7. H 2 0 8lμl 8. 40 cycles x [1' at 94°C; 3' at 37°C and 4' at 72°C] To analyze the PCR amplification products, lOμl of the 100 μl reaction were loaded onto a 1% agarose gel. Non-amplified controls and size markers were also included. Three distinct bands of about 350bp, 450bp and 700bp were observed. The three bands were blotted onto nitrocellulose paper and then hybridized to a synthetic radiolabeled DNA probe (probe C) corresponding to N-terminal amino acids 14 to 19 of the NH 2 -terminal sequence disclosed in Example 2 (section 2.1 above) and having the following sequence: A T T A T T T C: 5' - GA TG GA GA GG AA A - 3' C C G C G C C The three PCR products were found to hybridize with probe C under hybridization conditions of high stringency. However, the band corresponding to the 700 bp fragment was found to hybridize relatively poorly to the 350 bp and 450 bp fragments. Following PCR amplification, the DNA was purified from the reaction mixture by chloroform and phenol extractions and ethanol precipitation. The DNA was then digested with EcoRI and Hindlll and following gel purification, the fragments were subcloned into the large EcoRI-Hindlll fragment cf plasmid pSP65 (Figure 4) . The ligation mixture was used to transform E. coli strain MC1061. Transformants were screened by in-situ hybridization using the radiolabeled synthetic probe C disclosed above. Plasmid DNA was prepared from positive clones. Those plasmids containing an EcoRI and Hindlll fragment of the expected size were subjected to DNA sequencing using the Sanger dideoxy sequencing method. In this fashion two classes of clones were obtained: (A) those with an insert of about 290 base pairs (clones 3, 8 and 12) and (B) those with an insert of about 450 base pairs (clones 1, 4, 5 and 16) . The plasmids of clones 3 and 4 were designated pSP65- XaI-3 and pSP65-XaI-4 respectively. The DNA sequence and deduced amino acid sequence of clone pSP65-XaI-4 (clone 4) is shown in Figure 5. In-vitro translation using wheat germ lysate showed that both classes of clones encode a protein of the same size. It might therefore be that the shorter nucleotide sequence (290 bp) of clone 3 is due to internal priming by a poly-A rich sequence at the 3' non-translating region of the mRNA encoded by DNA of clone 4 which was used as template in the reverse transcription reaction described at the beginning of this Example. Example 4: Isolation and Cloning of DNA Encoding FXal From a Hirudo medicinalis cDNA library The PCR derived cDNA disclosed in Example 3 (clones 3 and 4) was isolated by hybridization with a probe corresponding to the N-terminal sequence of the purified naturally occurring protein. The DNA of clone 4 was then used as a probe to screen a cDNA library of Hirudo medicinalis. The procedure is shown schematically in Figure 6. Total RNA was extracted from 120 leeches. Poly A + mRNA was isolated from total RNA using the Fast Track™ mRNA isolation kit. An aliquot (4 μg) of the poly A + mRNA so obtained was used for ds-cDNA (double stranded cDNA) synthesis using the ZAP™ cDNA synthesis kit (Stratagene) . The resulting cDNA was subcloned using the Stratagene Predigested Lambda Zap ® II/EcoRI Cloning Kit. This kit which makes use of known methods of cloning provides for subcloning of the cDNA into lambda phage and amplification in E. coli. The phage is then isolated and the phagemid is excised with a helper phage. The resulting plasmids may be used for further manipulation by standard methods. 20ng of the resulting ds-cDNA was digested with Xhol and EcoRI and subcloned into the Xhol and EcoRI digested Uni- ZAP™ vector. Upon plating of the resulting recombinant phage on E. coli XLl-Blue™, a cDNA library of about 1.5 x 10 5 plaques was obtained (Figure 6) . This cDNA library was screened for clones containing DNA encoding FXa inhibitor using as probe radiolabeled DNA from the PCR derived plasmid, pSP65-XaI-4 (Example 3) under hybridization conditions of both low and high stringency. The prehybridization of the filter was carried out for 8 hours at 60°C in 6X SSC, 0.1% SDS, 5X Denhardt and 100 μg/ l salmon sperm DNA, followed by hybridization with the radioactive probe for 48 hours at 60°C. Filters were washed at two different conditions: to obtain high stringency conditions, filters were washed with 0.2-0.5 x SSC containing 0.2% SDS at 65°C for two hours with several changes of solution; to obtain low stringency conditions, filters were washed with 2X SSC-0.2% SDS for 2 hours at 60°C several times before autoradiography. Positive plaques were picked, isolated and rescreened under the same hybridization conditions. Plasmid DNA of several positive clones was then prepared and sequenced by the Sanger dideoxy method. The plasmid of one of the clones, obtained at low stringency, was designated pSK-XaI-13 (herein "clone 13" or "13") and selected for further manipulation and analysis. Other clones having different sequences were also obtained. Clone 13 contains a nucleotide sequence substantially different from that of the PCR derived clone 4 and encodes a correspondingly different protein. The amino acid sequence encoded by clones 4 and 13 are compared in Figure 3. The identification of clone 13 DNA by hybridization with clone 4 DNA was probably due to homology in the region of the predicted active site. Clone 13 encodes a novel polypeptide of 155 amino acids (not including the N-terminal methionine) which contains 27 cysteine residues, five of which are located in a 22 amino acid putative extension peptide. The polypeptide product of clone 13 is herein designated "FXal". The recombinant FXal product of clone 13 is also termed Yagin. The amino acid sequence of FXal is thought to encompass a mature protein and a putative extension peptide. The putative extension peptide is so called because although it does not appear to be part of the mature protein (based on the alignment of cysteine residues of FXal and other FXa inhibitors as disclosed below in Example 5: Alignment, and shown in Figure 3) , it does not have a composition typical of leader peptides, as discussed below. We analyzed the N-terminal amino acid sequence of FXal (Figure 7) for the presence of a putative leader sequence. The analysis was carried out using the algorithm of Von Heijne (Nucleic Acids Res. 14.: 4683-91, 1986) . This algorithm predicts the probability of a given amino acid to occupy a certain position within a leader sequence and thus can determine whether the peptide sequence conforms with that of a leader sequence. Moreover, by determining the "combined amino acid score" along the peptide, the cleavage site between the leader sequence and the mature protein can also be predicted. When applied to the analysis of the N- terminal sequence of the product of plasmid pSP65-XaI-13, this algorithm failed to identify a pattern of amino acids consistent with that of a leader peptide and no cleavage site was found within the first 40 residues. Based on the above analysis, it can be concluded that the protein encoded by plasmid pSP65-XaI-13 lacks a leader sequence. Thus, the sequence, beginning with met 1 and ending with cys 22 or glu 23 , appears to be an extension peptide and the sequence beginning with glu 23 or val :4 and ending with lys 1*56 appears to be the* mature protein. To be more specific, the polypeptide encoded by clone 13, as shown in Figure 7 extends from met 1 - Lys 156 . The segment extending from glu 23 or val 24 through cys 109 may constitute the functional -unit since it appears to be similar, including alignment of all the cysteines, to the sequences tyr 26 - gly 110 of clone 4, and cys 10 - cys 94 of antistasin, as shown in Figure 3. The nature of the extension peptide containing the first 22 amino acids encoded by clone 13 is not presently known, and has therefore been designated as a putative extension peptide. In order to confirm the existence of an open reading frame in clone 13, and to verify the size of the encoded polypeptide, the size of the polypeptide was determined using in vitro translation. SP6 RNA was prepared using the Stratagene in-vitro translation kit. The RNA thus obtained was subjected to in-vitro translation and the polypeptide products analyzed on SDS-PAGE under reducing conditions. The polypeptide produced by in-vitro translation of mRNA encoded by clone 13 migrated on the gel at a position corresponding to about 20kD (which is identical to the molecular weight of FXal expressed in E. coli) . This observed molecular weight is considerably higher than the molecular weight calculated from the amino acid sequence. Without wishing to be bound by theory, it is postulated that this may in part be due to the numerous cysteines present in the sequence of the clone 13 polypeptides which can affect the migration of the protein on the gel. Example 5: Comparison of deduced amino acid sequences The deduced amino acid sequences of the PCR-derived FXa inhibitor cDNA, FXal cDNA (clone 13 including the putative extension peptide) and antistasin were compared in different fashions i.e. maximum homology, identical homology, and alignment of cysteines. I. Homology Table 1 shows the degree of maximum homology of polypeptides (per cent) found using the 2020 PROSIS program (LKB) , Version 6.00. Maximum homology is based on Needleman and Wunsch, J. Mol. Biol. 4_8:443 ( 197 o) using the equivalence groups noted in the footnote to Table 1 and was determined on the full sequences including the putative extension peptides. Table 1 Degree of maximum homology * Clone Antistasin Clone 4 Clone 13 Antistasin 100 50 48 Clone 4 50 100 42 Clone 13 48 42 100 10 "Maximum homology was determined using the following equivalence groups: (1) A, S, T, P, G (2) N, D, E, Q (3) H, R, K 15 (4) M, L, I, V (5) F, W, Y Table 2 shows the degree of identical homology (expressed as per cent) which is the homology obtained without equivalence groups. Table 2 Degree of identical homology * CLONE Antistasin Clone 4 Clone 13 Antistasin 100 32 32 Clone 4 32 100 29 Clone 13 32 29 100 'Homology determined without equivalence groups In the degrees of homology determined above, the complete sequence of the polypeptide encoded by clone 13 including the "extension" peptide, was taken into account. II. Alignment Another way to analyze the similarity of sequences is to align the cysteines in the sequence, even at the expense of homology. The computer program Pileup based on the work of Feng and Doolittle, J. Mol. Evol. 315:351-360 (1987) performs such alignment. The resulting alignment utilizing the parameters of gap weight 3.00 and gap length weight 0.10, for the same sequences compared above, is shown in Figure 3. Figure 3 shows that 14 cysteines can be aligned without introducing considerable gaps in any of the sequences. The Pileup program assigns a value on a scale of 0-1.5 expressing the degree of similarity of sequences. The resulting degree of similarity of the present sequences is shown in Table 3 as a percent of 1.5. Table 3 Index of similarity Clone Clone 4 Clone 13 Antistasin Clone 4 100 52 41 Clone 13 52 100 34 Antistasin 41 34 100 10 It is thus seen that there is only little similarity between antistasin and the products of clone 4 and clone 13. This is shown both by comparison according to the degree of homology and according to the index of similarity. Example 6: Expression of Recombinant FXal In order to obtain plasmids for the expression of recombinant FXal in E. coli. further manipulation of the DNA fragments coding for the FXal protein was required. Plasmid pSK-XaI-13 (Example 4) was digested with Xhol and EcoRI. The Xhol-EcoRI fragment containing FXal was isolated and subcloned into a Sail-EcoRI digest of plasmid pSP65. The resulting plasmid was designated pSP65-XaI-13 (Figure 8) and deposited in the ATCC under Accession No. 69134. This plasmid does not express the encoded protein; however, it may be used in the construction of expression plasmids. By way of illustration, bacterial expression of recombinant FXal encoded by clone 13 was obtained by using cDNA fragments to construct various plasmids. A) Expression of mature ("m") recombinant FXal under control of λP L or deo promoters In order to enable the expression of the mature ("m") FXal polypeptide encoded by clone 13, an Ndel site containing the initiation codon ATG was introduced into the cDNA sequence adjacent to amino acid Val 24 by site directed mutagenesis using the following procedure: An aliquot of plasmid pSP65-XaI-13 was digested with EcoRI and Clal which cleaves within the FXal coding region. A second aliquot of the same plasmid was digested with XmnI and Seal which cleaves within the Amp R coding region. B'rom both aliqαots, the vector DNA was isolated, denatured and annealed in the presence of oligomer D Val D: 5' - GAATTGCGAAGCATATGGTAATAGCAG - 3' Ndel followed by reaction with DNA polymerase Klenow fragment and ligation using T 4 DNA ligase (Figure 9) . The ligation mixture was used to transform E. coli MC1061. Clones resistant to ampicillin and positive in hybridization to radiolabeled oligomer D were identified. One such clone was designated pSP65-XaI-13"m" . Plasmid pSP65-XaI-13"m" is not an expression plasmid. In order to obtain expression, suitable regulatory elements were provided as follows. Plasmid pSP65-XaI-13"m" was digested with Ndel and Hindlll. The Ndel-Hindlll fragment coding for the FXal polypeptide was isolated and ligated to the large fragment isolated from Ndel-Hindlll digestion of the λP L expression vector pMAL-183/191 which contains the λP L and λcll ribosomal binding site. The resulting plasmid was designated pλ-XaI-13"m" (Figure 10) and deposited in the ATCC under Accession No. 69135. Plasmid pλXaI-13"m" was used to transform E. coli 4300 which contains the thermolabile repressor cl 857 . Upon induction at 42°C, the transformed cells were found to express * a polypeptide having a molecular weight of about ~20kD. The sequence of this polypeptide was designed to be that of clone 13 in Figure 7 starting at Val 24 through Lys 156 . A similar plasmid could be made by one skilled in the art by using plasmid pMLK-100 which has been deposited in E. coli 4300 under ATCC Accession No. 68605 which is similar to plasmid pMAL-183/191 in the regions containing the regulatory elements. Furthermore, one skilled in the art knows how to construct similar expression plasmids using other known and readily available vectors. B) Expression of recombinant FXal as a Fused Protein with 58 N-Terminal Amino Acids of Human Cu/Zn-SOD Plasmid pSP65-XaI-13 was digested with Sspl and Pstl. The Sspl-Pstl fragment encoding FXal was isolated and ligated to the large fragment obtained from Seal-Pstl digestion of the SOD expression vector pBAST-R controlled by the deo promoter. The resulting plasmid was designated pDeo-S-Xal- 13"f" (Figure 11) and deposited in E. coli strain 733 in the ATCC under Accession No. 69137. Plasmid pDeo-S-XaI-13"f" encodes a fusion protein containing a 58 amino acid fragment of a modified Cu/Zn-SOD sequence and the FXal sequence of He 29 through Lys' 56 shown in Figure 7. The modified Cu/Zn-SOD fragment has the following sequence: MET ALA THR LYS ALA ALA SER VAL LEU LYS GLY ASP GLY PRO VAL GLN GLY ILE ILE ASN PHE GLU GLN LYS GLU SER ASP GLY PRO VAL LYS VAL TRP GLY SER ILE LYS GLY LEU THR GLU GLY LEU HIS GLY PHE HIS VAL HIS GLU PHE GLY ASP ASN THR ALA GLY SER Plasmid pDeo-S-XaI-13"f" was used to transform E. coli strain 733. A fused SOD-FXal recombinant protein having a molecular weight of about 2lkD was obtained in the insoluble fraction following lysis of the cell pellet from a culture of the transformed cells. C) Expression of FXal as a Prepeptide Containing the Predicted Extension Peptide In order to facilitate the expression of FXal, it was thought that inclusion of the putative extension peptide might enhance expression. Therefore, an Ndel site was introduced at Met 3 (Figure 7) using PCR. For PCR site directed mutagenesis the following 5' and 3' end primers were used: 5' end primer I 5' - CCGAATTCATATGTGTTGGAACAAAGGT - 3' Ndel 3' end primer J 5' - CCAAGCTTGGGCTGCAGGTCGA - 3' Hindlll Plasmid pSP65-XaI-13 was processed by PCR amplification in the presence of primers I and J. The product was then digested with Ndel and Hindlll. The fragment encoding FXal was isolated and ligated to the large fragment obtained from Ndel-Hindlll digestion of the λP L expression vector pMAL 183/191. The resulting plasmid was designated pλ-XaI-13"1" (Figure 12) . Plasmid pλ-XaI-13"l" was used to transform E. coli strain 4300. Following growth and induction of the transformed cells, FXal was obtained from inclusion bodies found in the insoluble fraction after lysis of the cell pellet. The sequence of this polypeptide was designed to be that of the product of clone 13 in Figure 7 including the extension peptide, starting at Lys 2 through Lys 156 . It is not yet known if the N-terminal methionine (met 1 ) is present. A similar plasmid could be made by one skilled in the art by using plasmid pMLK-100 which has been deposited in E. coli 4300 under ATCC Accession No. 68605 which is similar to plasmid pMAL-183/191 in the region containing the regulatory elements. Furthermore, one skilled in the art knows how to construct similar expression plasmids using other known and readily available vectors. Example 7: Refolding and partial purification of recombinant FXal produced by plasmid pλ-Xal- 13"m" A culture of E. coli 4300 containing plasmid pλ-XaI-13"m" was grown and induced essentially according to standard procedures known to those skilled in the art such as are disclosed in European Patent Publication No. 173,280, published March 5, 1986. The culture was grown at 30°C until mid-log phase and then induced at 42°C for 2 hours. The cells were pelleted and the cake stored frozen until processing. Isolation of Inclusion Bodies The bacterial cake was suspended in 10 volumes of buffer 1 (50mM Tris-HCl pH 8 containing lOmM EDTA) . Cell disruption was performed by adding lysozyme (2500U/ml) to the suspension and incubating with occasional stirring for 2 hours at room temperature. The suspension was then subjected to sonication (3x10 min) , and centrifugation (12,000 rpm) for 30 min at 4°C. The supernatant was discarded and the pellet washed with detergent by resuspension in 10 volumes of buffer 1 containing 1% Nonidet P-40 (NP-40) and incubation with occasional stirring for l hour at room temperature. The pellet which was obtained by centrifugation (12,000 rpm) for 30 minutes at 4°C was further washed with 10 volumes of distilled water, incubated with occasional stirring for 15 minutes at room temperature, and centrifuged (15,000 rpm) for 45 min at 4°C. The washing of the pellet resulted in removal of many of the E. coli proteins. It was established by SDS-PAGE that the recombinant FXal polypeptide remained in the pellet under the conditions of washing. 2. Solubilization and reduction Most of the FXal polypeptide was found in the inclusion bodies, i.e., in the insoluble pellet obtained following cell disruption. The inclusion bodies were then solubilized in 20 volumes of 6M guanidinium chloride (GuCl) in buffer 2 (20mM Tris-HCl pH 8 containing ImM EDTA and lOOmM NaCl) . Reduction was initiated after 15 min by the addition of 10 mM reduced glutathione (GSH) under a stream of nitrogen and was allowed to proceed for 1 hour. The protein concentration was then adjusted to 1 mg/ml by the addition of buffer 2 containing GSH and further incubated for 1-3 hrs under nitrogen. 3. Refolding/reoxidation The solubilized polypeptide so obtained which contains reduced FXal was diluted up to ' ten-fold in buffer 2 (final GuCl and GSH concentrations 0.6M and 1 mM, respectively) to a protein concentration of lOOμg/ml protein. Oxidized glutathione (GSSG) was added to a final concentration of 0.ImM and the solution was incubated for 16h at 4°C. The oxidized protein was dialyzed with 3 buffer changes against buffer 2 lacking EDTA, before assaying its inhibitory activity in the chromogenic assay described in Example 2. 4. Inhibitory activity of various batches of refolded recombinant FXal Following dialysis, the refolded FXal polypeptide was assayed for FXa inhibitory activity in the chromogenic assay described in Example 2. The results expressed in terms of milliunits (mu) per mg protein (mu/mg) are summarized in Table 4. The amount of protein causing 50% inhibition in an assay mixture containing 2nM FXa is defined as lmu. Table 4 Inhibition of FXa by Recombinant FXal * Refolded 13" " batch LG6X LG4X LG8X LG5X- 1 160 267 179 41 'Specific activity, mu/mg 10 ** Only partially refolded In addition, the refolded polypeptide was also tested for its effect on thrombin, another coagulation cascade enzyme, which was assayed according to Lottenberg et al. Methods Enzy ol. JLQ:341-361 (1981). No significant level of inhibition of thrombin by FXal was observed (<lmu/mg) . In summary, the recombinant FXal produced by the methods disclosed herein is biologically active. Furthermore, FXal has been demonstrated to be selective towards FXa, since the inhibitory activity of FXal towards FXa is approximately 200 fold greater than towards thrombin. Example 8: Elicitation of Antibodies Against Naturally Occurring FXa Inhibitor, and Recombinant FXal Antibodies were raised both against the SOD-FXal fusion polypeptide described in Example 6, and against a naturally occurring FXa inhibitor isolated from DLS. A. Preparation of antigens 1. Recombinant FXal fusion protein Antigen for immunization was prepared by expression of plasmid pDeo-S-XaI-13"f" (Example 6) , in E. coli 733 grown overnight at 37°C in LB containing 12.5 μg/ml tetracycline. The bacteria were pelleted by centrifugation, resuspended in 50 mM Tris-HCl pH 8 containing 1 mM EDTA and 50 μg/ml lysosyme, incubated for 30 minutes at room temperature, and sonicated twice for 5 minutes each time to reduce viscosity. Insoluble inclusion bodies containing the recombinant protein were collected by centrifugation. The pellet was resuspended in H : 0 and diluted with an equal volume of SDS- PAGE sample buffer (30% glycerol, 0.19M Tris-HCl pH 6.8, 10% SDS, 15% β -mercaptoethanol, and 0.004% bromphenol blue). The sample was then electrophoresed on a 12% SDS-PAGE gel, electro- ransferred onto a nitrocellulose membrane, and stained with the reversible Ponceau stain. Protein having an apparent molecular weight of 29 kD, was extracted from the membrane and used as antigen for immunization. 2. Naturally occurring FXa inhibitor from DLS Antigen for immunization was purified from DLS by Q- Sepharose and Heparin-Sepharose chromatography as described in Example 1. An aliquot of the protein was diluted with an equal volume of SDS-PAGE sample buffer (30% glycerol, 0.19M Tris-HCl pH 6.8, 10% SDS, 15% -mercaptoethanol, and 0.004% bromphenol blue) . The sample was then electrophoresed on a 15% SDS-PAGE gel, electro-transferred onto a nitrocellulose membrane, and stained with reversible Ponceau stain. Protein having an apparent molecular weight of about 14kD was extracted from the membrane, and used as antigen fcr immunization. B. Elicitation of antibodies Purified recombinant or naturally occurring antigen (20- 60μg) , dissolved in 1ml DMSO and mixed with complete Freund's adjuvant, was injected into the foot pads of a 1.8- 2.3kg rabbit. After 3 weeks, the rabbit was again injected, this time subcutaneously with the same amount of antigen mixed with incomplete Freund's adjuvant. This procedure was repeated twice more at the same interval. In order to remove anti-SOD antibodies, expected to be present in the antiserum to the recombinant SOD-containing FXal fusion protein, the antiserum was passed through an affinity column containing recombinant human Cu/Zn-SOD bound, to Sepharose. C. Antibody Verification by Western Blot Analysis Antibodies were verified on a Western blot of various proteins run on a 12% SDS-PAGE run under reducing conditions. Following reaction with the antisera, the Western blots were developed with 12J I-labeled protein A. D. Results Anti-13"f" antibody reacted with the 13"f" polypeptide and with the 13"m" polypeptide, but not with a recombinant protein having the amino acid sequence of the naturally occurring FXa inhibitor isolated from DLS. Furthermore, antibodies against naturally occurring FXa inhibitor reacted with naturally occurring FXa inhibitor (isolated from DLS) , but not with 13"m" (refolded and partially purified) . E. Conclusions We have obtained two anti-sera: the purified anti-13"f" which contains antibodies against the 13"m" recombinant polypeptide derived from clone 13, and antiserum to a naturally occurring FXa inhibitor isolated from DLS. Based on the results with both antisera, we can deduce that there is no immunological cross-reaction between the naturally occurring FXa inhibitor isolated from DLS and the recombinant 13"m" FXal polypeptide. Therefore, these results clearly show that clone 13 encodes a protein which is substantially different from the naturally occurring FXal protein, present in DLS. F. Identification of naturally occurring FXal in H. medicinalis Since clone 13 was obtained from a cDNA library, it would reasonably be expected that the encoded protein should be present in DLS. However, using the anti-13"f" antibodies described above, no band was detected, even in the presence of a very high protein amount (70μg) . Since no protein was detected in DLS by the assays described above, this novel FXal may not be present in leech saliva. This would account for its not being observed in the course of purification of DLS. However, the presence of a protein band with a molecular weight of about 20 kD which reacted on a Western blot with antibodies to the FXal recombinant protein was demonstrated in supernatants of homogenized sections of the leech body. This confirmed the presence of the protein in the leech corresponding to the product of clone 13 (Yagin) . Exampl 9: Purification and Biological Activity of Refolded FXal Recombinant FXal expressed by plasmid pλ-XaI-13"m", produced and refolded essentially as described in Example 7 was purified to homogeneity and assayed for biological activity. The refolded protein was purified by ultrafiltration on a 50K cutoff membrane, and further processed by Heparin- Sepharose and Q-Sepharose chromatography using NaCl gradients in lOmM Tris-HCl pH 8. The activity peak eluted from Heparin-Sepharose at about 300mM NaCl and was then loaded onto Q-Sepharose. The activity peak eluted from the Q-Sepharose at about lOOmM NaCl. The purity of the protein so obtained was verified on SDS- • PAGE under reducing conditions where it showed an apparent molecular weight of 20kD, and on Superose 12 gel filtration chromatography where it eluted with a retention time equivalent to about 15kD. The purified FXal had biochemical activity of about 2500 mu/mg in the chromogenic assay described in Example 2. The K, was determined to be about 13nM in the chromogenic assay and l60pM in the prothrombinase complex assay described in Example 2. The IC J0 was determined to be 20-40nM in the chromogenic assay and about 540pM in the prothrombinase assay. APTT (activated partial thromboplastin time) of the purified protein was measured in human and murine plasma essentially as described in Spaethe, Haemostasis. AHS/Deutschland GmbH, Munich (1984) , using as reagent Actin FS (Dade) , which contains ellagic acid for activation of Factors XII and Factor XI, and soy bean phospholipid. Under these conditions, and in the presence of 20mM CaCl 2 , only the intrinsic pathway of coagulation is activated. The results showed that 23nM (343 ng/ml) caused a doubling of the APTT in human plasma. In comparison, 50nM heparin was required in the same assay to obtain the doubling of the APTT. Biological experiments: Pharmacokmetic Study of FXal A preliminary pharmacokmetic experiment was performed in order to assess the clearance rate of FXal from the circulatory system. Female Balb/c mice (20-25 gm body weight) were injected intravenously with purified FXal. Blood samples were drawn into 1/10 v/v 3.8% citrate at 1, 3, 10 and 30 min after injection. The plasma was then separated and assayed for APTT. It was possible to obtain only one blood sample from each mouse and therefore each timepoint is from a different mouse. FXal concentration was calculated from a calibration curve of APTT versus FXal concentration which was constructed in vitro by adding various amounts of FXal to a plasma pool from untreated mice. In this manner, the blood half-life was determined to be about 8.1 minutes. Example 10: Mature FXal Polypeptide Obtained from a Cleavable Fusion Protein Low levels of expression of the mature FXal polypeptide were obtained from plasmid pλ-XaI-13"m" (Figure 10) as described in Example 6. Although plasmid pDeo-S-Xal-13"f" (Figure 11) produced significant quantities of an FXal fusion polypeptide as described in Example 6, it was not possible to obtain the 13"m" polypeptide, since there was no cleavage site between the SOD moiety and the FXal moiety. In order to obtain larger quantities of the 13"m" polypeptide, a plasmid expressing a cleavable fusion polypeptide was constructed. I. Construction of plasmid pλSOD-NGE-XaI-13"m" The following procedures are shown schematically in Figures 13 and 2. Plasmid pλ-XaI-13"l" (Figure 12) was digested with Sspl. The fragment encoding FXal was isolated, further digested with Aatll, and the large fragment isolated. Plasmid pBAST- R (Figure 11) was digested with Aatll and PpuMI and the large fragment isolated. The Aatll-Sspl fragment containing the FXal-encoding DNA was then ligated to the large pBAST-R fragment in the presence of the following two synthetic oligomers: AsnGly Oligomer K: 5' - GTCCTAATGGTGAAGTAATAGCAGAGAAT - 3' Oligomer L: 3' - GATTACCACTTCATTATCGTCTCTTA - 5' The resulting plasmid, designated pDSOD-NGE-XaI-13"m" ( Figure 13) , under control of the deo promoter, was used to transform E. coli 733, but was determined to be a poor expressor of the FXal fusion protein. In an attempt to obtain improved expression, a plasmid for expression of the FXal fusion protein under control of the thermolabile λ P L promoter was constructed. Plasmid pDSOD- NGE-XaI-13"m" was digested with Ndel and Hindlll. The small fragment containing the FXal coding region was isolated and ligated to the large fragment isolated from Ndel - Hindlll digestion of plasmid pλAB (Figure 2) . The resulting plasmid, designated pλS0D-NGE-XaI-13"m" (Figure 2), under control of the λP L promoter, was used to transform E. coli 4300 (F) , and was determined to be a good expressor of the FXal fusion polypeptide. Plasmid pλSOD-NGE-XaI-13"m" encodes in 5' to 3' order a fusion protein containing a 63 amino acid fragment of a modified Cu/Zn-SOD sequence, three amino acids Asn-Gly-Glu (containing the hydroxylamine cleavage site Asn-Gly) , and the FXal 13"m" polypeptide having the sequence val 24 -lys 156 shown in Figure 7. The SOD moiety has the following amino acid sequence: MET ALA THR LYS ALA ALA SER VAL LEU LYS GLY ASP GLY PRO VAL GLN GLY ILE ILE ASN PHE GLU GLN LYS GLU SER ASP GLY PRO VAL LYS VAL TRP GLY SER ILE LYS GLY LEU THR GLU GLY LEU HIS GLY PHE HIS VAL HIS GLU PHE GLY ASP ASN THR ALA GLY SER THR SER ALA GLY PRO. II. Production of the 13"m" polypeptide from plasmid pλSOD- NGE-XaI-13"m" E. coli 4300(F) containing plasmid pλSOD-NGE-XaI-13"m" was grown at 30°C till mid-log phase and then induced for two hours at 42°C. The FXal fusion protein accumulated in inclusion bodies, which were isolated and washed as described in Example 7. Washed inclusion bodies were solubilized in 20 volumes of nitrogen-flushed buffer 3 (20mM Tris-HCl pH 8, 50mM NaCl, ImM EDTA, and 20mM GSH) containing 5.5M guanidinium thiocyanate (GuSCN) . Following solubilization, the fusion protein was cleaved by incubation for 16 hours at room temperature at a concentration of 0.7mg/ml in IM hydroxylamine pH 9 (adjusted with LiOH) also containing 1.75M GuCl, 2.75M GuSCN, lOmM Tris, 25mM NaCl, 0.5M EDTA, and lOmM GSH. After incubation, the solution was dialyzed against buffer 3 containing 3.5M GuCl. The FXal was then refolded by incubation at a concentration of O.lmg/ml in buffer 3 lacking GSH but containing 0.3mM GSSG. The crude refolded protein so obtained inhibited FXa with specific activity of 150-300 mU/mg as determined by the chromogenic assay. Example 11: Production of Recombinant FXal in Eukaryotic Cells Due to the inherent complexity of refolding a molecule containing many cysteines, such as FXal, to obtain a biologically active polypeptide, it is postulated that production of the polypeptide in eucaryotic cells might eliminate of some of the difficulties. This would enable production of large amounts of properly refolded, biologically active recombinant protein. In preliminary experiments, expression of the mature 13"m" FXal polypeptide was obtained using a baculovirus expression system in Sf-9 insect cells (18) . The cells had been transformed with DNA encoding the 13"1" protein, i.e. DNA encoding the FXal polypeptide and its extension sequence (as contained in plasmid PSK-XaI-13 (Figure 6)). Western blot analysis showed that the resulting polypeptide accumulated in the insect cells and was neither processed nor secreted into the medium, consistent with the lack of a leader peptide (enabling secretion) at the N-terminus of the polypeptide (Example 4) . The polypeptide cross-reacted with antibodies to recombinant FXal produced as described in Example 8. Example 12: Improved expression plasmids and growth conditions for FXal A ^ Plasmid pλ-Xal-13"m"-T I T ? In order to eliminate overproduction of β-lactamase driven by the strong lambda P L promoter, a T^ transcription termination DNA fragment was introduced into plasmid pλ-XaI-13"m" (Figure 10) resulting in plasmid pλ-XaI-13"m"- T.T 2 (Figure 26) . Plasmid pλ-XaI-13"m"-T,T 2 was used to transform E. coli 4300, which contains the thermolabile repressor cl 857 . Upon induction at 42°C, the transformed cells were found to express a polypeptide having a molecular weight of about 20kD at an expression level similar to that obtained with plasmid pλ-XaI-13"m" . However, since there is no overproduction of β-lactamase protein, the percentage of protein which is FXal in plasmid pλ-XaI-13"m"-T,T 2 is higher than the percentage of protein which is FXal in plasmid pλ- Xal-13"m" . ΪL. Plasmid pλ-XaI-13"m"-05M-T,T ? Plasmid pλ-Xal-13"m"-05M (Figure 14) was constructed in order to both improve the level of expression of FXal and introduce an extra amino terminal amino acid, glycine, adjacent to the initiation codon ATG (encoding Met) , which facilitates complete removal of the Met in the host bacteria following expression. The newly introduced Met-Gly is situated immediately before Val 24 in Figure 7. In order to eliminate overproduction of β-lactamase driven by the strong lambda P L promoter, a T j T 2 transcription termination DNA fragment was introduced into plasmid pλ-XaI-13"m"-05M resulting in plasmid pλ-XaI-13 l, m"-05M-T,T (Figure 15) . Plasmid pλ-XaI-13"m"-05M-T ι T 2 was used to transform E. coli 4300, which contains the thermolabile repressor cl 857 . Upo induction at 42°C, the transformed cells were found t express a polypeptide having a molecular weight of about 20kD at an expression level two to three times higher tha that of plasmid pλ-XaI-13"m" . C. Improved conditions for bacterial growth and Inductio The level of expression of FXal from plasmids pλ-XaI-13"m"- T,T 2 and pλ-XaI-13"m"-05M-T,T 2 was low when using 1 x L supplemented with 0.1% glucose as growth medium. To optimize growth and induction, a series of experiment was carried out. It was found that when using 5 x L supplemented with 0.1% glycerol as the growth medium an when changing the temperature of heat induction to 39°C fo 4 hours (instead of 42°C) , the level of expression of FXa is several fold higher. Example 13: Improved production of pure and active FXal Refolding and purification of FXal expressed by plasmid pλ- XaI-13"m"-TιT 2 described in Example 12 was carried out as described in Examples 7 and 9 apart from the following changes in order to improve the production of FXal. Processing was scaled-up and the purity of the processed inclusion bodies was increased to 40% as evaluated by SDS- PAGE. Refolding and purification were scaled-up as shown in Scheme l. The concentration of reducing agent, GSH, was increased from 10 mM to 20mM. The Heparin-Sepharose column was replaced by a S-Sepharose column. Concentration and dialysis were scaled-up and were performed on an ultrafiltration system, such as Pellicon (Millipore) or Production Ultrafiltration (PUF, Millipore) with a lOkDa cutoff membrane, allowing completion of those steps within about 5h. In addition some other minor changes were made to the FXal production protocol and all the improvements were incorporated into the improved production protocol, which is described in Scheme 1. Scheme 1: FXal expressed by plasmid pλ-Xal-13"m" -T t T : PROCESSING, REFOLDING AND PURIFICATION STEP BACTERIAL Processing Lysozyme digestion CAKE Gaulin homogenizer, centrifugation; NP-40, centrifugation; water, centrifugation INCLUSION Dissolution, 16L 6M GuCl, 20mM GSH, BODIES reduction Buffer A. IH, RT REDUCED Refolding, 160L 0.6M GuCl, 2mM PROTEIN reoxidation GSH, 0.2mM GSSG, Buffer A, 72H, 4°C, 50mcg/mL Concentration, Pellicon (lOkDa) to dialysis, 2.5L; dialysis lOmM filtration Tris-HCl (pH8) ; filtration Cuno 30 REFOLDED, Purification (1) Q-Sepharose lOmM ACTIVE FXal Tris-HCl (pH8) ; elution +250mM NaCl (2) S-Sepharose 20mM acetate (pH5) ; elution +400mM NaCl PURE, ACTIVE FXal in PBS Buffer A: 20mM Tris-HCl (pH8) , 50mM NaCl, ImM EDTA PBS: 10 mM Na-phosphate (pH 7.4), 150 mM NaCl In summary, from 1.5 gram of bacterial cake, about 180 mg of FXal (>95% pure) was produced. Example 14: Characterization of FXal FXal (produced as described in Example 13) was characterized as described below. FXal is a protein containing 133 amino acids beginning with val (val 24 in Figure 7) with calculated molecular weight 15.4 kD. FXal contains 22 cysteine residues, which are probably paired in 11 disulfide bonds. Biochemical and stability properties FXal is a homogeneous protein, more than 95% pure as evaluated by SDS-PAGE under reducing conditions (Scheme 1) . FXal contains less than 1% dimers and aggregates as determined by gel filtration on Superose 12. The endotoxin levels as determined by the gel clot technique using Limulus amelocyte lysate (LAL) reagent (Yin, E.T. et al. (1972), Biochem. Biophys. Acta. 261: 284-289) , are less than 4 EU/mg. FXal is stable in a lyophilized state, as well as in frozen form in PBS (preferably at -70°C) at a protein concentration of l-2mg/ml (Bradford-SDS) . 2. Inhibitory activity of FXal FXal inhibits FXa and trypsin, but does not inhibit thrombin and plasmin. (i) Inhibition of FXa The FXa inhibitory activity of FXal was tested in two assays: (a) Inhibition of soluble FXa (15,000 mU/mg) was measured in an in vi tro chromogenic assay (as described in Example 2) . In this assay 1 mu is equivalent to the amount of material causing 50% inhibition in the assay, which contains 2nM active FXa. The results of the assay are presented in Table 5 below. It is clear from this Table that FXal is a high affinity inhibitor of FXa. (b) Thrombin generation in the reconstituted prothrombinase complex assay (as described in Example 2) was inhibited as is shown in Table 5 below. TABLE 5: FXal inhibition of FXa in the chromogenic and reconstituted prothrombinase complex assays Ki, nm BATCH OF FXal CHROMOGENIC ASSAY PROTHROMBINASE ASSAY 1 7.2 0.060 2 10.8 0.045 3 14.0 0.028 (ii) Inhibition of Trypsin Inhibition of bovine trypsin by FXal was calculated following the classical dose response method. Ki values were obtained by plotting the percentage of activity of FXal (determined in the colorimetric assay described below) against the ratio of the concentrations of inhibitor and enzyme. The colorimetric assay was performed using 80 μM Chro ozyme TH (Pentapharm, Switzerland) as substrate in 10 M Tris-HCl, 10 mM Hepes (pH 7.8), 100 mM NaCl, 0.1% PEG 6000 at 25°C in presence of 3.83 or 4.27 nM of bovine trypsin. Inhibitor and enzyme were preincubated for 10 min at 25°C before adding the substrate. In this assay, the Ki values for two batches of FXal were 7.6 and 6.0 nM. (iii) Inhibition of Plasmin and Thrombin Plasmin and thrombin were not inhibited by FXal in a similar assay as that described above for trypsin. In the case of thrombin, the substrate used was the same as that used for trypsin; in the case of plasmin, S2251 (KabiVitrum, Sweden) was used as substrate. 3. Antibodies Antibodies were raised against pure FXal produced as described in Example 13. (i) Elicitation of antibodies Both refolded, intact FXal (1.9 mg/ml) in phosphate buffered saline (PBS) and denatured FXal (applied to 15% acrylamide SDS-PAGE transferred to nitrocellulose and solubilized in 0.5 ml DMSO) , were injected into rabbits. Antigen (800 μg/ml) in PBS was mixed with an equal volume (o.5 ml) of Ribi adjuvant (Ribi adjuvant system - RAS - Ribi ImmunoCohem Research, Inc, Hamilton, MT, USA) , whereas the DMSO solubilized antigen was injected into rabbit with the Ribi adjuvant without mixing. The antigen (1 ml) was administered to rabbits (2.5-4.5 kg) as follows: 0.05 ml intradermally in six sites; 0.2 ml intramuscularly into each hind leg; 0.1 ml subcutaneously in the neck region; and 0.2 ml intraperitoneally. Rabbits were injected every 28 days and test bled 10 days after injection. Booster injections were repeated ten times. (ii) Antibody verification by Western Blot Analysis Specificity of the antibodies was verified on a Western Blot of specific and unrelated proteins, run on a 12% SDS-PAGE gel under reducing conditions. Following reaction with antisera, a specifc reaction was detected using a non- radioactive light-based system (ECL system, Amersham International P/C, Amersham, England) . (iii) Results The antibody against the refolded FXal recognized both the refolded and denatured forms of FXal, whereas the antibody against the reduced FXal recognized reduced FXal only. Neither antibody recognized unrelated proteins. Example 15: Biological activity of FXal The experiments were performed with two batches of FXa produced as described in Example 13. I. In vi tro APTT FXal-APTT dose response curves were constructed using plasm from man, mouse, rabbit, rat and baboon. FXa concentrations of l-3.3μg/ml (Table 6), equivalent to 70-25 nM, caused a doubling of the APTT. Using rat plasma, th APTT doubling concentrations of heparin and low molecula weight heparin (LMWH) were 1.3 and 3.3 IU/ml, equivalent t 8.1 and 13.2 μg/ml, respectively. II. Pharmacokinetics and pharmacodynamics of FXal in variou animals As described below, pharmacokinetics and pharmacodynamics o FXal were evaluated in mice, rats, rabbits and baboons. Th pharmacokinetics were based on the rate of disappearance o radiolabeled FXal from the plasma of the various animals, whereas pharmacodynamics were based on the time-dependen decrease of the ex vivo APTT. Pharmacokinetics FXal was iodinated ( 125 I) by reaction with the Bolton-Hunte reagent (Bolton and Hunter (1973) , Biochem. J. 133: 529 539) , admixed with unlabeled material (specific activit 0.13 x IO 6 cpm/μg) and injected into mice (10 μg/mouse) Analysis of plasma samples during 3h revealed that th labeled material was fully TCA precipitable at all times an its level declined with a half-life (t-^) of 41 minutes Within 5 minutes after administration, about 40% of th radioactivity was found in the liver. The amount in th liver then decreased gradually .in parallel with the amount in the plasma. Concomitantly, 125 I-labeled material accumulated in the intestines, thus indicating that FXal was probably degraded proteolytically in the liver, followed by excretion of the degraded products into the intestines. Labeled FXal was additionally injected into rats and rabbits. In rats, the half-life of plasma radioactivity was about 76 min and in rabbits it was about 94 min (Figure 16) . (ii) Pharmacodynamics It was found that upon FXal administration, ex vivo APTT is prolonged, relative to saline or PBS controls, in all animals studied. The APTT values decline in a time- dependent manner as FXal is being cleared. These pharmacodynamic results are summarized in Table 6. The pharmacodynamics of FXal were additionally studied following i.p., s.c. and i.m. administration to mice. In these three routes of administration, the maximum APTT was achieved between 60-90 min, and the bioavailability was between 35-50%. TABLE 6: FXal concentration causing APTT doubling and biological Half-Life in Various Species Species APTT Doubling Half- Concentration life (μg/ml) (min) Mouse 1.2 78 Rat 2.2 61-92 Rabbit 1.7 70-78 Baboon 3.3 120 Human 1.0 ND III. Anti-thrombotic activity in venous thrombosis model The stainless steel coil-induced venous thrombus model in rats was used to study the inhibitory effect of FXal on venous thrombus formation. The model employed was as described by Maffrand et al. [Thrombosis and Haemostasis 59: 225-230 (1988)]. Wistar-derived female rats (200-250 g) were anaesthetized by Ketamine HC1 plus Xylazin HC1. A midline abdominal incision was made and the inferior vena cava was exposed. A stainless steel wire coil (a dental paste carrier, fine No. 31, 21 mm long) was inserted into the lumen of the vein, at the site just below the junction, and the incision was sutured. Each inserted device was individually weighed before insertion and each weight was recorded. Subsequently, the rats were sacrificed. The segment of the vein carrying the coil was removed; the vein sections were incised longitudinally, and the coils carrying the thrombi were carefully removed and weighed. The initial weight of each coil was subtracted from its final weight. Using this model, the anti-thrombotic activity of FXal was compared with that of heparin. Mature clot formation within 2-3 hours is characteristic of this model. Hence, pharmacodynamic studies were performed using multiple i.v. injection regimens in order to attain a continuous plasma level of the anti-thrombotic agents. Plasma levels were estimated by ex vivo APTT measurements. It was found that satisfactory plasma profiles (± 20% fluctuations) were obtained with both heparin and FXal by administration of an initial i.v. dose at 0-time, followed by two doses of 35% of the initial dose at 45 min intervals (Figure 18) . Using various dose levels in this regimen, clot formation was examined by insertion of the coils 5 min after the initial injection and clot measurements were taken 45 min after th third injection. ID50 is the dose of FXal causing 50 decrease in the amount of clot formed. The anti-thromboti effect seen with 2 doses of FXal (compared to a PBS control) is shown in Figure 19. These results were compared to th anti-thrombotic effect of heparin in this model, and ar summarized in Table 7 below. IV. Effect of FXal and heparin on tail bleeding in mice Bleeding time was measured in pentobarbitone anesthetize mice. Bleeding was induced by standardized trans-section o the tips of the tails, and subaqueous bleeding time wa determined after immediate vertical immersion of the tail in isotonic saline at 37°C. This comparative study was done in order to evaluate th adverse effect, i.e., bleeding, of both FXal and heparin. T assess this effect, tail bleeding time was determined i mice injected i.v. with FXal and heparin, 5 min afte injection. The prolongation of the bleeding time, i.e., th increase over control, for FXal and heparin are shown i Figures 20 and 21 respectively. V. Beneficial versus risk effects of FXal and heparin. Table 7 shows the doses required for a twofold prolongatio of the bleeding time, as well as the ID50 values in th venous thrombosis model, for FXal and heparin. The rati between these two values is a direct measure of the relativ benefit versus risk effects. For FXal, this calculated rati is 2.6, whereas heparin gave a value which was lower by on order of magnitude (0.2) . In conclusion, FXal was found t be a highly effective anti-thrombotic agent, with a superio benefit/risk ratio over that of heparin. TABLE 7: Anti-thrombotic and bleeding parameters of FXal and heparin AGENT ANTI-THROMBOSIS (RAT BLEEDING BENEFIT COIL MODEL) TIME: /RISK TWOFOLD RATIO ID50 (a) AVERAGE PROLONGATION (c) APTT DOSE (a) (sec) (b) FXal 67 μg/kg 41 176 μg/kg 2.6 Heparin 80 IU/kg 63 17 IU/kg 0.2 (=500μg/kg) (=106μg/kg) (a) Initial injected dose (b) Ex-vivo APTT for the injected dose (control APTT = 29 sec) (c) Ratio of the bleeding time doubling dose to the ID50 Exa ple 16: Inhibition of Influenza Virus Propagation in Fertilized Chicken Eggs bv FXal Establishment of the pharmacokinetic behavior of FXal in fertilized chicken eggs The pharmacokinetic behavior of FXal, produced as described in Example 13, in fertilized chicken eggs was determined by the time-dependent decrease of the ex vivo APTT. FXal (300 μg/egg) was injected into the chorioallantoic fluid (CAF) of fertilized chicken eggs. Aliquots of the CAF were removed at various time points and assayed for APTT prolongation in rat citrated plasma (3.8%, v/v 1:10) . APTT prolongation activity was found to decrease by first-order kinetics, with a half-life of 24 hours. This finding served as a basis for adopting a 4-hour interval injection regimen in the virus propagation study. Effect of FXal on influenza virus propagation in fertilized eggs Influenza virus type A (PR-8) was injected (10" 2 EID 50 in 0.1 ml saline per egg) into fertilized eggs. The eggs were divided into 3 groups. One group (Group A) was injected with FXal (20μg/egg) in 0.2ml saline on 0-time, then lOμg/egg (0.1ml) at 4, 8, 12 and 16h post-injection. The second group (Group B) received FXal (20μg/egg) at 0-time, and another injection of lOμg/egg at 4 h. The third group received 0.2ml saline at 0-time followed by 0.1ml saline at 4, 8, 12 and 16h, and served as a control. The eggs were maintained in a rolling mode at 37°C. At 24, 32 and 48h post-injection, 4-10 eggs were taken from each group, the CAF removed and the levels of influenza virus examined in a hemagglutination assay (19) . The results are presented in Table 8. TABLE 8: Effect of FXal on influenza virus titer in CAF of fertilized chicken eggs VIRUS TITER (log 2 dilution) GROUP 24h 32h 48h Control 5,3,4,0,0 0,8,5,7,8 6,6,6,6 Group A 0,0,0,0,0, 0,0,0,0,0 6,8,5,7,8 (higher dose) 0,0,0,0,0 Group B 0,0,0,0,0 0,5,4,8,7 8,8,8,8 (lower dose) The results indicate that FXal inhibits the propagation of the influenza virus in chicken embryonic cells. In addition, these results indicate that FXal inhibits influenza virus propagation in eukaryotes and will treat or prevent infection of subjects suffering from influenza virus or at risk of contracting influenza. Example 17: Inhibition of acute arterial thrombosis by FXal in the rat The inhibitory effect of FXal on acute arterial thrombosis was tested in an acute arterial thrombosis model in the rat. The model deployed was as decribed by Bernat, A., et al. [Thromb. Haemostas., ZQ, 812-816 (1993)] with slight modifications as described below. Thrombus formation was induced by electrical stimulation of the carotid artery in rats. Wistar-derived male rats (250- 350 g) were anaesthetized with Ketamine-HCl plus Xylazin-HCl and placed on a heated blanket. A segment of the right carotid artery (about 10 mm-long) was exposed and dissected free of surrounding tissue. A small piece of insulating film (Parafilm) and two stainless steel electrodes were inserted under the artery. Using a constant d.c. power supply, the artery was stimulated at 0.98 mA during 4 min. The thrombotic occlusion was monitored by measuring the local artery temperature cranially (downstream) from the site of electrical stimulation. Both body and arterial temperatures were measured at 20 min intervals over a 60 min period. An I.V. injection (0.5 ml) of saline (in control animals) or FXal (at various concentrations) was administered to the rats 5 min before the onset of electrical stimulation. The FXal batch used in these experiments was produced as described in Example 13. However, even though this batch of FXal had essentially the same activity in the chromogenic assay as previous batches of FXal, it displayed only about 65% of their potency in the APTT assay, i.e., the concentration of FXal causing doubling of the APTT was 50% higher than in the previous batches of FXal. The results for control animals (n=12) and three distinct doses of FXal (0.133 mg/kg (n=7) , 0.266 mg/kg (n=9) and 0.4 mg/kg (n=4)) are shown in Figure 22. The results indicate that both higher doses of FXal cause total abolition of the arterial thrombosis-associated temperature decrease, whereas the lowest dose displays a partial effect. Example 18: Inhibition of thrombosis by FXal in baboons Inhibition of thrombus formation in baboons by FXal produced as described in Example 13, was measured under conditions of variable blood shear rate using a dacron thrombogenic device that generates platelet-rich and fibrin-rich thrombi essentially as described in inter alia Cadroy et al. , Blood 75(11) : 2185-2193 (1990) , Kelly et al . (1991) , Blood 27: 1006-1012 and Cadroy et al. (1991), P.N.A.S. ___S 1177-1181 with some slight modifications as shown in Figure 25. Three baboons were each injected with three distinct doses of FXal: 10, 40 and 200 μg/kg/hr. Half of the total dose was given as a bolus and the other half infused over one hour. The baboons were injected with πι In-platelets and their accumulation was measured by a gamma camera. Steady state plasma levels were: undetectable at the lowest dose, 0.6 μg/ml at the intermediate dose, and 3.6 μg/ml at the high dose. The half-life of FXal in the plasma of baboons is about two hours. Platelet deposition on the Dacron graft, representative of arterial thrombosis, was reduced by an average of 30%, 50% and 80% respectively by these doses (Figure 23) . In addition, each of these doses nearly abolished thrombus formation in the "Chamber" region of low-shear venous-type flow, representative of venous thrombosis (Figure 24) . Furthermore, one baboon was injected with PBS and served as a control. In this baboon there was maximal level of arterial and venous platelet accumulation. The results indicate that FXal inhibits both venous thrombosis and arterial thrombosis in baboons. Moreover, the effect of FXal on venous thrombosis can already be seen at the lowest concentration of FXal (10 μg/kg/hr) . In addition, the amount of FXal, needed to nearly abolish dacron arterial platelet deposition in baboons, is about two order's of magnitude smaller than the amount of desulfato- hirudin needed to obtain such an effect (Kelly et al. (1991), Blood 77: 1006-1012, especially Figure 1C, 20nmol desulfato-hirudin/kg/min ≡ 7 mg hirudin/kg/hour) . The amount of FXal needed to abolish venous platelet deposition in baboons is also about two orders of magnitude smaller than the amount of H-peptide (Hirulog) needed to obtain such an effect (Cadroy et al.(1991), P.N.A.S. 18: 1177-1181, especially Figure 1A) . Furthermore, the amount of FXal, needed to nearly abolish dacron arterial platelet deposition in baboons, is about a fifth of the amount of Tick Anticoagulant Peptide (TAP) needed to obtain such an effect (Kelly et al. , 65 th scientific sessions of the American Heart Association, New Orleans, Louisiana, USA, November 16-19, 1992. Circulation 86 (4 suppl. 1) 1992, 1-411.) Ref erences 1. Tuszynski et al., J. Biol. Chem 262:9718-9723 (1987) 2. EPO Publication No. 263,608, published April 13, 1988, of Gasic et al, assigned to The Contributors to the Pennsylvania Hospital 3. Condra et al . , Thro b. Haemostas. 61:437-441 (1989) 4. U.S. Patent No. 4,832,849, May 23, 1989, of Cardin, assigned to Merril Dow Pharmaceuticals 5. European Patent Application Publication No. 419,099, Vlasuk, et al . , March 27, 1991 6. Teitel et al. , J. Clin. Invest. 21:1383-1391 (1983) 7. Dunwiddie et al . , Thromb. Haemostas. £7:371-376 (1992) 8. Dunwiddie et al. , J. Biol. Chem. 264:16694-16699 (1989) 9. Waxman et al . , Science 211:593-596 (1990) 10. Neeper et al., J. Biol. Chem. 26_5:17746- (1990) 11. Jacobs, Thromb. Haemostas. 64 . :235-238 (1990) 12. Vlasuk et al., Thromb. Haemostas. 65:257-269 (1991) 13. U.S. Patent No. 5,196,335, issued May 23, 1993, Rigbi et al . assigned to Yissum Research Development Co. 14. Hirsh, Drug Therapy 324:1565-1574 (1991) 15. Gold, N. Eng. J. of Med. 321:1483-1485 (1990) 16. Weitz, et al. , J. Clin. Invest. 11:385-391 (1990) 17. Sitko, et al., Circulation 15:805-815 (1992) 18. Neutra, et al. , Appl. Microbiol. Biotechnol 37:74-78 (1992) 19. Purchare et al. (1987),. A laboratory manual for the isolation and identification of avian pathogens, Third Edition, American Association of Avian Pathalogists. 194.

Claims: What is claimed is; 1. A polypeptide comprising the amino acid sequence X - Y - CYS GLN GLU GLU GLU CYS PRO ASP PRO TYR LEU CYS SER PRO VAL THR ASN ARG CYS GLU CYS THR PRO VAL LEU CYS ARG MET TYR CYS LYS PHE TRP ALA LYS ASP GLU LYS GLY CYS GLU ILE CYS LYS CYS GLU GLU LEU CYS GLN ASN GLN ASN CYS THR LYS ASP MET LEU CYS SER SER VAL THR ASN ARG CYS AS CYS GLN ASP PHE LYS CYS PRO GLN SER TYR CYS - Z wherein X is MET or absent; Y is 0-29 amino acids of the sequence LYS MET CYS TR ASN LYS GLY CYS PRO CYS GLY GLN ARG CYS ASN LEU HIS AR ASN GLU CYS GLU VAL ILE ALA GLU ASN ILE GLU, with th proviso that if part of the sequence is present, it is the carboxy part of the sequence and wherein Val 24 ma be preceded by Gly; and Z is absent or all or a part of the sequence Pro 110 - Lys 156 shown in Figure 7, with th proviso that if part of the sequence is present, it i the amino part of the sequence. 2. A polypeptide according to claim 1 having the amin acid sequence Y-Lys 2 -Lys 156 where Lys 2 -Lys 156 is identica to the sequence shown in Figure 7 and wherein Y is ME or absent. 3. A polypeptide according to claim 1 comprising the amin acid sequence Y-lie 29 -Lys 156 where Ile 29 -Lys 156 is identica to the sequence shown in Figure 7 and wherein Y is ME or absent. 4. A polypeptide according to claim 1 having the amin acid sequence Y-Val 24 -Lys 156 where Val^-Lys 156 is identica to the sequence shown in Figure 7, and wherein Y is MET or absent and wherein Val 24 may be preceded by Gly. 5. A non-glycosylated polypeptide of any of claims 1-4. 5 6. DNA encoding the polypeptide of claim 1. 7. DNA encoding the polypeptide of claim 4. 10 8. A plasmid comprising the DNA of claim 6 designated pSP65-FXaI-13 and deposited under ATCC Accession No. 69134. 9. An expression plasmid comprising the DNA of claim 6. 15 10. An expression plasmid of claim 9 designated pλ-Xal- 13"m" deposited under ATCC Accession 69135. 11. An expression plasmid of claim 9 designated pλ-Xal- 20 13 n m"-T,T 2 . 12. An expression plasmid of claim 9 designated pDeo-S-Xal- 13 "f" deposited under ATCC Accession No. 69137. 25 13. An expression plasmid comprising the DNA of claim 7. 14. An expression plasmid of claim 13 designated pλ-Xal- 13"m"-05M. 30 15. An expression plasmid of claim 13 designated pλ-Xal- 13"m"-05M-T,T 2 . 16. A host-plasmid system comprising the expression plasmid of claim 9, 10, 11, 12, 13, 14 or 15 in a host cell. ■ 35 17. A host-plasmid system of claim 16 wherein the host cel is a bacterial cell. 18. A host-plasmid system of claim 17 wherein the bacteria cell is an E. coli cell. 19. A method of producing the polypeptide of claim l whic comprises transforming a host cell with an expressio plasmid encoding the polypeptide, culturing th transformed host cell so that the cell produces th polypeptide encoded by the plasmid, and recovering th polypeptide so produced. 20. A method according to claim 19 wherein the host cell i a bacterial cell and the recovering comprises: (a) disrupting the cells so as to produce a lysat containing the polypeptide; (b) treating the lysate so as to obtain inclusio bodies containing the polypeptide; (c) treating the inclusion bodies so as to obtain th polypeptide in soluble form; (d) treating the resulting soluble polypeptide so a to form biologically active polypeptide; (e) recovering the biologically active polypeptide s formed; and (f) purifying the biologically active polypeptide s recovered. 21. A method of claim 20 wherein the treating of step (c) comprises the addition of a denaturant. 22. A method of claim 21 wherein the denaturant is guanidinium chloride or urea. 23. A method of claim 20 wherein the treating of step (d) comprises contacting the polypeptide with a mixture of a thiol-containing compound and a disulfide. 24. A method of claim 23 wherein the thiol containing compound is glutathione, thioredoxin, β - mercaptoethanol, or cysteine and the disulfide is oxidized glutathione, cystine, or the product of air oxidation of mercaptoethanol. 25. A method of claim 20 wherein the purifying of step (f) comprises column chromatography. 26. A method of claim 25 wherein the column chromatography comprises either one or both of Q-Sepharose chromatography and Heparin-Sepharose chromatography or either one or both of Q-Sepharose chromatography and S- Sepharose chromatography. 27. A composition comprising the polypeptide of any of claims 1-4 and 52 in an amount effective to obtain a desired therapeutic effect resulting from the biological activity of the polypeptide, and a suitable carrier. 28. A composition of claim 27 wherein the desired therapeutic effect comprises the reduction of the extent of blood coagulation. 29. A method of reducing the extent of blood coagulation comprising contacting the blood with an amount of the polypeptide of claim 1 effective to reduce the extent of blood coagulation. 30. A method of claim 29 wherein the contacting is effected in vivo in a subject. 31. A method of claim 30 wherein the subject suffers from excessive blood coagulation. 32. A method of claim 31 wherein the subject suffering from excessive blood coagulation has a condition selected from the group consisting of vascular disorders, post- operative trauma, obesity, pregnancy, side affects of oral contraceptives, and prolonged immobilization. 33. A method of claim 32 wherein the excessive blood coagulation comprises a cerebrovascular disorder. 34. A method of claim 33 wherein the cerebrovascular disorder is stroke. 35. A method of claim 31 wherein the excessive blood coagulation comprises a thrombosis. 36. A method of claim 35 wherein the thrombosis is a venous thrombosis. 37. A method of claim 36 wherein the venous thrombosis is deep venous thrombosis. 38. A method of claim 36 wherein the venous thrombosis is disseminated intravascular coagulation. 39'. A method of claim 35 wherein the thrombosis is an arterial thrombosis. 40. A method of claim 39 wherein the arterial thrombosis is thrombosis of a coronary artery . 41. A method of claim 35 wherein the thrombosis occurs following thrombolysis. 42. A method of claim 41 wherein the thrombolysis is effected with a fibrinolytic agent. 43. A method of claim 42 wherein the fibrinolytic agent is tissue plasminogen activator or streptokinase. 44. A method of claim 42 wherein the polypeptide is administered together with the fibrinolytic agent. 45. A method of claim 42 wherein the polypeptide is administered before or after the fibrinolytic agent. 46. A method of claim 42 wherein the polypeptide is bound to the fibrinolytic agent. 47. A method of inhibiting the activity of Factor Xa which comprises contacting Factor Xa with an amount of the polypeptide of claim 1 effective to inhibit the activity of the Factor Xa. 48. An antibody which specifically reacts with an epitope of the polypeptide of claim 1. 49. A monoclonal antibody according to claim 48. 50. A polypeptide which competitively inhibits the specific reacting of the antibody of claim 48. . 51. An expression plasmid of claim 9 designated pλSOD-NGE- Xal-13"m" deposited under ATCC Accession No. 69269. 52. A polypeptide according to claim 1 having the amino acid sequence Glu 23 -Lys 1*56 where Glu 23 -Lys 1*56 is identical to the sequence shown in Figure 7. 53. A non-glycosylated polypeptide of claim 52. 54. A method of treating or preventing influenza comprising administering to a subject suffering from influenza or at risk of contracting influenza, an amount of an inhibitor of FXa effective to treat or prevent influenza. 55. A method of claim 54 wherein the inhibitor of FXa is administered in conjunction with superoxide dismutase. 56. A method of treating or preventing influenza comprising administering to a subject suffering from influenza or at risk of contracting influenza, an amount of the polypeptide of claim l or 4 effective to treat or prevent influenza. 57. A method of claim 56 wherein the polypeptide is administered in conjunction with superoxide dismutase. 58. A polypeptide of claim 1, 2, 3, 4 or 5 having Factor Xa inhibitory activity.

Inventors: LEVANON AVIGDOR, WERBER MOSHE M, ZEELON ELISHA P, LEVANON, AVIGDOR, WERBER, MOSHE, M., ZEELON, ELISHA, P.
Applicants: BIO TECHNOLOGY GENERAL CORP, BIO-TECHNOLOGY GENERAL CORP.
Application year: 1994
Main classification: A61K37
IPC codes: A61K38, C07K14, C12N1, C12R1